Random-primed transcriptase in-vitro transcription method for rna amplification

ABSTRACT

A random-primed reverse transcriptase-in vitro transcription method of linearly amplifying RNA is provided. According to the methods of the invention, source RNA (or other single-stranded nucleic acid), preferably, mRNA, is converted to double-stranded cDNA using two random primers, one of which comprises a RNA polymerase promoter sequence (“promoter-primer”), to yield a double-stranded cDNA that comprises a RNA polymerase promoter that is recognized by a RNA polymerase. Preferably, the primer for first-strand cDNA synthesis is a promoter-primer and the primer for second-strand cDNA synthesis is not a promoter-primer. The double-stranded cDNA is then transcribed into RNA by the RNA polymerase, optimally in the presence of a reverse transcriptase that is rendered incapable of RNA-dependent DNA polymerase activity during this transcription step. The subject methods produce linearly amplified RNA with little or no 3′ bias in the sequences of the nucleic acid population amplified.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of, and claimspriority to U.S. application Ser. No. 13/896,872 filed May 17, 2013,which is a continuation of application Ser. No. 13/372,320 filed on Feb.13, 2012; which is a continuation of U.S. application Ser. No.12/790,234 filed on May 28, 2010 (abandoned); which is a continuation ofU.S. application Ser. No. 11/745,386 filed on May 7, 2007 (abandoned),which is a division of U.S. application Ser. No. 10/432,176 filed onNov. 13, 2003 (now U.S. Pat. No. 7,229,765), which is a §371 filing ofInternational Application No. PCT/US01/44821 filed on Nov. 28, 2001,which application claims the benefit of U.S. Provisional Application No.60/253,641 filed on Nov. 28, 2000. Said applications are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to enzymatic amplification of nucleicacids using two random primers, one of which contains a RNA polymerasepromoter sequence, to generate a double stranded DNA template, and invitro transcription.

BACKGROUND OF THE INVENTION

The characterization of cellular gene expression finds application in avariety of disciplines, such as in the analysis of differentialexpression between different tissue types, different stages of cellulargrowth or between normal and diseased states. Recently, changes in geneexpression have also been used to assess the activity of new drugcandidates and to identify new targets for drug development. The latterobjective is accomplished by correlating the expression of a gene orgenes known to be affected by a particular drug with the expressionprofile of other genes of unknown function when exposed to that samedrug; genes of unknown function that exhibit the same pattern ofregulation, or signature, in response to the drug are likely torepresent novel targets for pharmaceutical development. One particularlyuseful method of assaying gene expression at the level of transcriptionemploys DNA microarrays (Ramsay, Nature Biotechnol. 16: 40-44, 1998;Marshall and Hodgson, Nature Biotechnol. 16: 27-31, 1998; Lashkari etal., Proc. Natl. Acad. Sci. (USA) 94: 130-157, 1997; DeRisi et al.,Science 278: 680-6, 1997).

A number of methods for the amplification of nucleic acids have beendescribed. Such methods include the “polymerase chain reaction” (PCR)(Mullis et al., U.S. Pat. No. 4,683,195), and a number oftranscription-based amplification methods (Malek et al., U.S. Pat. No.5,130,238; Kacian and Fultz, U.S. Pat. No. 5,399,491; Burg et al., U.S.Pat. No. 5,437,990). Each of these methods uses primer-dependent nucleicacid synthesis to generate a DNA or RNA product, which serves as atemplate for subsequent rounds of primer-dependent nucleic acidsynthesis. Each process uses (at least) two primer sequencescomplementary to different strands of a desired nucleic acid sequenceand results in an exponential increase in the number of copies of thetarget sequence. These amplification methods can provide enormousamplification (up to billion-fold). However, these methods havelimitations that make them not amenable for gene expression monitoringapplications. First, each process results in the specific amplificationof only the sequences that are bounded by the primer binding sites.Second, exponential amplification can introduce significant changes inthe relative amounts of specific target species-small differences in theyields of specific products (for example, due to differences in primerbinding efficiencies or enzyme processivity) become amplified with everysubsequent round of synthesis.

Amplification methods that utilize a primer containing a RNA polymerasepromoter sequence (“promoter-primer”) are amenable to the amplificationof heterogeneous mRNA populations. The vast majority of mRNAs carry ahomopolymer of 20-250 adenosine residues on their 3′ ends (the poly-Atail), and the use of poly-dT primers for cDNA synthesis is afundamental tool of molecular biology. “Single-primer amplification”protocols have been reported (see e.g., Kacian et al., U.S. Pat. No.5,554,516; Van Gelder et al., U.S. Pat. No. 5,716,785). The methodsreported in these patents utilize a single promoter-primer containing aRNA polymerase promoter sequence and a sequence complementary to the3′-end of the desired nucleic acid target sequence(s). In both methods,the promoter-primer is added under conditions in which it hybridizes tothe target sequence(s) and is converted to a substrate for RNApolymerase. In both methods, the substrate intermediate is recognized byRNA polymerase, which produces multiple copies of RNA complementary tothe target sequence(s) (“antisense RNA”). Each method uses, or could beadapted to use, a primer containing poly-dT for amplification ofheterogeneous mRNA populations.

Amplification methods that proceed linearly during the course of theamplification reaction are less likely to introduce bias in the relativelevels of different mRNAs than those that proceed exponentially. In themethod described in Kacian et al., U.S. Pat. No. 5,554,516, theamplification reaction contains a nucleic acid target sequence, apromoter-primer, a RNA polymerase, a reverse transcriptase, and reagentand buffer conditions sufficient to allow amplification. Theamplification proceeds in a single tube under conditions of constanttemperature and ionic strength. Under these conditions, the antisenseRNA products of the reaction can serve as substrates for furtheramplification by non-specific priming and extension by the RNA-dependentDNA polymerase activity of reverse transcriptase. As such, theamplification described in U.S. Pat. No. 5,554,516 proceedsexponentially. In contrast, in specific examples described in Van Gelderet al., U.S. Pat. No. 5,716,785, cDNA synthesis and transcription occurin separation reactions separated by phenol/chloroform extraction andethanol precipitation (or dialysis), which may incidentally allow forthe amplification to proceed linearly since the RNA products cannotserve as substrates for further amplification.

The method described in U.S. Pat. No. 5,716,785 has been used to amplifycellular mRNA for gene expression monitoring (for example, R. N. VanGelder et al. (1990), Proc. Natl. Acad. Sci. USA 87, 1663; D. J.Lockhart et al. (1996), Nature Biotechnol. 14, 1675). However, thisprocedure is not readily amenable to high throughput processing. Inpreferred embodiments of the method described in U.S. Pat. No.5,716,785, poly-A mRNA is primed with a promoter-primer containingpoly-dT and converted into double-stranded cDNA using a method describedby Gubler and Hoffman (U. Gubler and B. J. Hoffman (1983), Gene 25,263-269) and popularized by commercially available kits for cDNAsynthesis. Using this method for cDNA synthesis, first strand synthesisis performed using reverse transcriptase and second strand cDNA issynthesized using RNaseH and DNA polymerase I. After phenol/chloroformextraction and dialysis, double-stranded cDNA is transcribed by RNApolymerase to yield antisense RNA product. The phenol/chloroformextractions and buffer exchanges required in this procedure are laborintensive, and are not readily amenable to robotic handling.

A method of linear amplification of mRNA into antisense RNA has beenrecently developed, U.S. Pat. No. 6,132,997 issued to Shannon(“Shannon”), which is incorporated by reference in its entirety for allpurposes. Shannon does not require a reverse transcriptase separationstep and is therefore readily amenable to high throughput processing.Shannon discloses a method in which mRNA is converted to cDNA(particularly double-stranded cDNA) using a promoter-primer having apoly-dT primer site linked to a promoter sequence so that the resultingcDNA is recognized by a RNA polymerase. The resultant cDNA is thentranscribed into RNA (particularly antisense RNA) in the presence of areverse transcriptase that is rendered incapable of RNA-dependent DNApolymerase activity during the transcription step.

A significant drawback of the Shannon method, however, is that itproduces a 3′ bias in the amplification of mRNA. Sequences that are morethan 1000 by from the 3′ end to which the primer has hybridized areunderamplified with respect to sequences that are less than 1000 by fromthe 3′ end, i.e., the sequences that are more than 1000 by from the 3′end are amplified in less than linear amounts.

Thus there exists a need in the art for an improved method of linearamplification of mRNA that is amenable to high throughput processing,that produces little or no 3′ bias, that improves the ability to detectthe 5′ ends of mRNA, and therefore achieves good representation of boththe 3′ and 5′ regions of an original mRNA in the amplified complementaryRNA (cRNA).

SUMMARY OF THE INVENTION

A random-primed reverse transcriptase-in vitro transcription method oflinearly amplifying RNA is provided. According to the methods of theinvention, source RNA (or other single-stranded nucleic acid),preferably, mRNA, is converted to double-stranded cDNA using two randomprimers, one of which comprises a RNA polymerase promoter sequence(“promoter-primer”), to yield a double-stranded cDNA that comprises aRNA polymerase promoter that is recognized by a RNA polymerase.Preferably, the primer for first-strand cDNA synthesis is apromoter-primer and the primer for second-strand cDNA synthesis is not apromoter-primer. The double-stranded cDNA is then transcribed into RNAby the RNA polymerase, optimally in the presence of a reversetranscriptase that is rendered incapable of RNA-dependent DNA polymeraseactivity during this transcription step. The subject methods ofproducing linearly amplified RNA provide an improvement over priormethods in that little or no 3′ bias in the sequences of the nucleicacid population amplified is produced, and the ability to detect the 5′end sequences of the nucleic acids is improved. The methods of theinvention therefore achieve good representation of both the 3′ and 5′regions of the source nucleic acid in the amplified complementary RNA(cRNA). Linear amplification extents of at least 100-fold can beachieved using the subject methods. All of the benefits of linearamplification are achieved with the subject methods, such as theproduction of unbiased antisense RNA libraries from heterogeneous mRNAmixtures.

In particular, the invention provides a method for linearly amplifyingone or more single stranded nucleic acids, said method comprising (a)contacting said one or more single stranded nucleic acids with a firstset of oligonucleotides, each of which comprises a promoter sequence anda sequence from a set of random sequences of at least 4 nucleotides (butpreferably 6 to 9 nucleotides, more preferably 9 nucleotides), a secondset of oligonucleotides, each of which comprises (preferably, consistsof) of one or a set of random sequences of at least 4 nucleotides (butpreferably 6 to 9 nucleotides, more preferably 6 nucleotides) and one ormore enzymes that alone or in combination catalyze the synthesis ofdouble-stranded cDNA, under conditions suitable for the production ofdouble-stranded cDNA; and (b) contacting the double-stranded cDNAproduced in step (a) with a RNA polymerase that recognizes said promotersequence and ribonucleotides under conditions suitable to effecttranscription, thereby producing sense or antisense RNA copiescorresponding to said one or more single stranded nucleic acids. In apreferred embodiment, the second set of oligonucleotides does notcontain a promoter sequence. Alternatively, the cDNA may be generated intwo steps where the first step is the synthesis of first strand cDNAusing the first set of oligonucleotides and one or more enzymes thatcatalyze first strand cDNA synthesis and the second step is thesynthesis of double-stranded cDNA by contacting the first strand cDNAmade in the first step with the second set of oligonucleotides and oneor more enzymes that alone or in combination catalyze second strand cDNAsynthesis. In preferred embodiments, the enzyme used in step (a) is areverse transcriptase. In an alternative embodiment, the single-strandednucleic acid is also contacted in step (a) with a promoter-primercontaining the same promoter sequence used in the set of randomprimer-promoter primers used in step (a) and a polydT sequence of atleast 4 nucleotides (preferably at least 5 nucleotides, more preferably15 to 25 nucleotides, and most preferably 18 nucleotides).

The invention further provides kits for carrying out the linearamplification methods of the invention containing one or more componentsused in the methods of the inventions and instructions for use. In aparticular embodiment, the invention provides a kit for use in linearlyamplifying single stranded nucleic acids into sense or antisense RNA,said kit comprising a first set of oligonucleotides each comprising apromoter sequence and one of a set of random sequences of at least 4nucleotides; and a second set of oligonucleotides each of whichcomprises (preferably, consists of) of one of a set of random sequencesof at least four nucleotides. In a preferred embodiment the second setof oligonucleotides does not contain a promoter sequence[.?] In anotherembodiment, the kit also contains a reverse transcriptase and a RNApolymerase. In yet another embodiment, the kit further contains, inaddition to the two sets of random primers, oligonucleotides containingthe same promoter sequence as the random primer-promoter primeroligonucleotide and a polydT sequence of at least 5 nucleotides(preferably 18 nucleotides).

DESCRIPTION OF THE FIGURES

FIGS. 1(A-B). Comparison of profiles obtained from single-gene analysisusing (A) the mRNA amplification method described in U.S. Pat. No.6,132,997 (Shannon, issued Oct. 17, 2000) (“Shannon”) and (B) therandom-primed reverse transcriptase-in vitro transcription (RT-IVT)method of the invention. The graphs plot signal intensity (mlavg) ofoligonucleotides in a single gene (X-axis) as a function of the numberof base pairs from the 5′ end (Y-axis). The 3′ bias of signal intensityseen when the Shannon method is used cannot be seen when therandom-primed RT-IVT method is used, indicating that the random-primedRT-IVT method overcomes the 3′ bias of the Shannon method.

FIG. 2. Intensity difference as a function of distance from the 3′ end.The graph shows the intensity of all oligonucleotides as a function ofdistance from the 3′ end. The graph plots mlavg (Shannon method) - mlavg(random-primed RT-IVT method) (X-axis) versus log₁₀ of the number of byfrom the 3′ end (Y-axis). The intensity obtained with the Shannon methodis greater than the intensity obtained with the random-primed RT-IVTmethod for probes less than 1000 by from the 3′ end of the message. Theintensity obtained with the Shannon method is less than the intensityobtained with the random-primed RT-IVT method for probes greater than1000 by from the 3′ end of the message.

FIGS. 3(A-C). Signature differences in the numbers and percentages ofsignificant data points. The top graph (A) plots the number of probes(X-axis) versus the log₁₀ (bp) (Y-axis). The middle graph (B) plots thenumber of signatures (X-axis) versus the log₁₀ (bp) (Y-axis). The bottomgraph (C) plots the fraction of signatures versus the log₁₀ (bp)(Y-axis). As can be seen in the bottom graph, the random-primed RT-IVTmethod outcompetes the Shannon method for probes greater than 1000 byfrom the 3′ end. Note the black arrow at approximately 700 by whererandom-primed RT-IVT method is more representative than the Shannonmethod. Stars: Shannon method. Circles: random-primed RT-IVT method.

FIGS. 4(A-C). Shows the results obtained when the amplification methodsof the invention were run using a primer comprising a T7 RNA polymerasepromoter site and an poly-dT₁₈ sequence (“T7-dT₁₈”), in addition tousing random T7-dN₉ and dN₆ primers. The top graph (A) plots the numberof probes (X-axis) versus the log₁₀, (bp) (Y-axis). The middle graph (B)plots the number of signatures (X-axis) versus the log₁₀ (bp) (Y-axis).The bottom graph (C) plots the fraction (“frac”) or percentage ofsignatures versus the log₁₀ (bp) (Y-axis). As can be seen in the bottomgraph, the number of probes at greater than 1000 base pairs is greaterwith the random-primed RT-IVT method. Using both the T7-dT₁₈ and randomT7-dN₉ primers for first strand cDNA synthesis improves the fraction ofstatistically significant probes more efficiently than either theShannon method or the method of the invention in which just the randomT7-dN₉ primer is used. Stars: Shannon method. Circles: random-primedRT-IVT method.

DETAILED DESCRIPTION OF THE INVENTION

A random-primed reverse transcriptase-in vitro transcription (RT-IVT)method of linearly amplifying RNA is provided. According to the methodsof the invention, source RNA (or other single-stranded nucleic acid),preferably, mRNA, is converted to double-stranded cDNA using two randomprimers, one of which comprises a RNA polymerase promoter sequence(“promoter-primer”), to yield a double-stranded cDNA that comprises aRNA polymerase promoter that is recognized by a RNA polymerase. Thus,“promoter sequence” refers to a single-stranded nucleotide sequence thatwhen double-stranded (i.e., paired with its reverse-complement) forms aRNA polymerase promoter that is recognized by a RNA polymerase.Preferably, the primer for first-strand cDNA synthesis is apromoter-primer and the primer for second-strand cDNA synthesis is not apromoter-primer. Optionally, the cDNA synthesis reaction contains amixture of the random-sequence-promoter primer and an oligonucleotidecontaining the promoter sequence and an oligodT sequence. Thedouble-stranded cDNA is then transcribed into RNA by the RNA polymerase,optimally in the presence of a reverse transcriptase that is renderedincapable of RNA-dependent DNA polymerase activity during thistranscription step.

The subject methods of producing linearly amplified RNA provide animprovement over prior methods in that little or no 3′ bias in thesequences of the nucleic acid population amplified is produced, and theability to detect the 5′ end sequences of the nucleic acids is improved.The methods of the invention therefore achieve good representation ofboth the 3′ and 5′ regions of the source nucleic acid in the amplifiedcomplementary RNA (cRNA). Linear amplification extents of at least100-fold can be achieved using the subject methods. All of the benefitsof linear amplification are achieved with the subject methods, such asthe production of unbiased antisense RNA libraries from heterogeneousmRNA mixtures.

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections set forthbelow.

5.1. Methods of Nucleic Acid Amplification

The invention provides methods for producing amplified amounts of eithersense or antisense RNA from an initial amount of source single-strandednucleic acid, preferably poly-A+ RNA or mRNA. By amplified amounts ismeant that for each initial source of nucleic acid, multiplecorresponding sense or antisense RNAs are produced. The term antisenseRNA is defined here as RNA complementary to the source single-strandednucleic acid. By corresponding is meant that the sense or antisense RNAshares a substantial sequence identity with the sequence of, or thesequence complementary to (i.e., the complement of the initial sourcenucleic acid), the source nucleic acid. Substantial sequence identitymeans at least 95%, usually at least 98%, and more usually at least 99%,and, in certain embodiments, 100% sequence identity, where sequenceidentity is determined Using the BLAST algorithm, as described inAltschul et al. (1990), J. Mol. Biol. 215:403-410 (using the publisheddefault setting, i.e., parameters w=4, t=17). Generally, the number ofcorresponding antisense RNA molecules produced for each initial nucleicacid during the subject linear amplification methods will be at leastabout 10, usually at least about 50, more usually at least about 100,and may be as great as 600 or greater, but often does not exceed about1000.

The subject methods can be used to produce amplified amounts of RNAcorresponding to substantially all of the nucleic acid present in theinitial sample, or to a proportion or fraction of the total number ofdistinct nucleic acids present in the initial sample. By substantiallyall of the nucleic acid present in the sample is meant more than 90%,usually more than 95%, where that portion not amplified is solely theresult of inefficiencies of the reaction and not intentionally excludedfrom amplification.

In a specific embodiment, only a single cycle of reverse transcriptionis carried out. In alternative embodiments, more than one cycle ofreverse transcription is performed (with transcription and denaturationbetween cycles). For example, in a specific embodiment, a first cycle ofreverse transcription is carried out wherein one or more single strandednucleic acids are (a) contacted with a first set of oligonucleotides,each of which comprises a promoter sequence and a sequence from a set ofrandom sequences of at least 4 nucleotides (but preferably 6 to 9nucleotides, more preferably 9 nucleotides), a second set ofoligonucleotides, each of which comprises (preferably, consists of) ofone or a set of random sequences of at least 4 nucleotides (butpreferably 6 to 9 nucleotides, more preferably 6 nucleotides) and one ormore enzymes that alone or in combination catalyze the synthesis ofdouble-stranded cDNA, under conditions suitable for the production ofdouble-stranded cDNA. The resultant double-stranded cDNA is then (b)contacted with a RNA polymerase that recognizes said promoter sequenceand ribonucleotides under conditions suitable to effect transcription(i.e., in vitro transcription or “IVT”), thereby producing sense orantisense RNA copies corresponding to said one or more single strandednucleic acids. The resultant sense or antisense RNA copies are thenreverse transcribed in a second cycle of reverse transcription, asdescribed in step (a) above, and the resultant double-stranded cDNA isthen transcribed via IVT into sense or antisense RNA copies as describedin step (b) above. Additional cycles of RT-IVT may be performed toobtain the desired quantity of sense or antisense RNA copies.

According to the methods of the invention, additional linearamplification is afforded by a subsequent in vitro transcription (IVT)step as described below in Section 5.2. During IVT, the double-strandedcDNA produced in the first step is transcribed by RNA polymerase toyield RNA that is complementary to the initial RNA target from which itis amplified. This combination of cDNA synthesis and IVT enables thegeneration of a relatively large amount of cRNA from a very smallstarting amount of nucleic acid without loss of fidelity, andparticularly, without 3′ amplification bias.

In one embodiment of the invention (see Example 1, Section 6), 0.2 μg(200 ng) of source mRNA is used.

In another embodiment of the invention, nucleic acid amplification isperformed in situ, on samples of preserved or fresh cells or tissues(see, e.g., Nuovo, 1997, PCR In situ Hybridization: Protocols andApplications, Third Edition, Lippincott-Raven Press, New York).

The subject methods may be applied to other amplification systems inwhich an oligonucleotide is incorporated into an amplification productsuch as polymerase chain reaction (PCR) systems (U.S. Pat. No.4,683,195, Mullis et al., entitled “Process for amplifying, detecting,and/or-cloning nucleic acid sequences,” issued Jul. 28, 1987; U.S. Pat.No. 4,683,202, Mullis, entitled “Process for amplifying nucleic acidsequences,” issued Jul. 28, 1987).

5.1.1. cDNA Synthesis

Double-stranded cDNA molecules can be synthesized from a collection ofRNAs (or other single-stranded nucleic acids), e.g., mRNAs present in apopulation of cells, by methods well-known in the art. In order for thecDNAs produced in this step to be useful in the methods of theinvention, it is necessary to incorporate a RNA polymerase promoter intothe cDNA molecules during synthesis. This enables the cDNA molecules toserve as templates for RNA transcription. This is accomplished bychoosing one or more primers for the cDNA synthesis reaction thatcomprise a single-stranded, synthetic oligonucleotide containing a RNApolymerase promoter sequence in sense orientation. This“promoter-primer” may be used to prime either first strand and/or secondstrand cDNA synthesis. In preferred embodiments, the “promoter-primer”primes first strand cDNA synthesis and the promoter is in theappropriate orientation to promote synthesis of antisense RNA.

Typically, only one RNA polymerase promoter sequence-containing primeris used during cDNA synthesis. Preferably, the promoter-primer is usedto prime first strand cDNA synthesis. Following reverse transcription,the resultant RNA polymerase promoter-containing double-stranded cDNA istranscribed into RNA using a RNA polymerase capable of binding to theRNA polymerase promoter introduced during cDNA synthesis (see belowSection 5.2).

In a preferred embodiment, the primer for first strand cDNA synthesis isa mixture of random primers linked to a promoter sequence that primesynthesis in a direction toward the 5′ end of the nucleic acids (e.g.,mRNAs) in the sample, and the primer for second strand cDNA synthesis isa mixture of random primers that prime synthesis of double-stranded cDNAfrom substantially all the first strand cDNAs thus produced.

Preferably, the first-strand primer is a random promoter-primer, whereinthe random (poly-dN) sequence is operably linked to a RNA polymerasepromoter sequence. In one aspect, the first-strand primer is a mixtureof primers, each primer comprising a RNA polymerase promoter sequenceand a 3′ end or 3′ distal sequence of 6-9 nucleotides, preferably 9nucleotides. The mixture of primers comprises random primers, i.e.,primers having an A, a G, a C, or a T residue present in each positionof the 3′ end sequence or 3′ distal sequence (i.e., the non-promotersequence). In particular, the random primer for priming first strandcDNA synthesis is a random promoter-primer that includes: (a) a poly-dNregion for hybridization to the mRNA; and (b) a RNA polymerase promoterregion 5′ of the poly-dN region that is in an orientation capable ofdirecting transcription of antisense RNA when it primes first strandcDNA synthesis. The poly-dN region is sufficiently long to provide forefficient hybridization to the mRNA, where the region typically rangesin length from 4-50 nucleotides in length, preferably 6-25 nucleotidesin length, more preferably from 6-12, and most preferably, 9 nucleotidesin length, i.e., a random 9-mer. In specific embodiments, the poly-dNregion is 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length.

In a preferred embodiment, the random promoter-primer used to primefirst strand cDNA synthesis is a random 9-mer operably linked to a T7RNA polymerase promoter sequence (T7-dN₉: (5′) AAT TAA TAC GAC TCA CTATAG GGA GAT NNN NNN NNN (3′) (N=A, T, C or G) (SEQ ID NO.: 1)).

In another embodiment, the random promoter primers used to prime firststrand cDNA synthesis are a complete set of all (or almost all)combinations of random 9-mers, i.e., a total of 4⁹ 9-mers, linked to aT7 RNA polymerase promoter sequence.

In another embodiment, a poly-dT primer comprising a RNA polymerasepromoter sequence and a random dN primer comprising a RNA polymerasepromoter sequence are used together to prime first strand cDNAsynthesis. Preferably, the poly-dT-promoter primer and the randomprimer-promoter primer contain the same promoter sequence. In particularembodiments the poly-dT sequence is at least 5 thymidilate residues,preferably 15 to 25 residues and, preferably 18 residues. In a preferredembodiment, a T7-dT₁₈ primer and a T7-dN₉ primer are used to prime firststrand cDNA synthesis.

A number of RNA polymerase promoters may be used for the promoter regionof the promoter-primer. Suitable promoter regions will be capable ofinitiating transcription from an operably linked DNA sequence in thepresence of ribonucleotides and a RNA polymerase under suitableconditions. The terns “operably linked” refers to a functional linkage,i.e., the promoter will be linked in an orientation to permittranscription of sense or antisense RNA. Preferably the linkage iscovalent, most preferably by a nucleotide bond. Most preferably, thepromoter is linked in an orientation to permit transcription ofantisense RNA when the promoter is incorporated into the first strand ofcDNA synthesis. A linker oligonucleotide between the promoter and theDNA may be present, and if,[<cut“,”?] present, will typically comprisebetween about 5 and 20 bases, but may be smaller or larger as desired.The promoter region is of sufficient length to promote transcription,and will usually comprise between about 15 and 250 nucleotides,preferably between about 17 and 60 nucleotides, from a naturallyoccurring RNA polymerase promoter or a consensus promoter region, asdescribed in Alberts et al. (1989) in Molecular Biology of the Cell, 2dEd. (Garland Publishing, Inc.), or any other variant that promotestranscription. In a specific embodiment, the promoter region is 36nucleotides. Preferred promoter regions include the bacteriophage SP6and T3 promoters and, most preferably, T7 promoters.

The random promoter-primer and/or the random primer may additionallycontain a restriction site, in the middle or at the 5′ distal end of theprimer, but preferably not immediately at the 5′ terminus. Therestriction site may be used for cloning in to a vector. Restrictionenzymes and the sites they recognize can be found, for example, inSambrook et al., 1989, Molecular Cloning—A Laboratory Manual (2nd Ed.),Vol. 1, Chapter 5, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

The primers of the invention may be prepared using any suitable methodknown in the art, e.g., as described in Section 5.3 infra.

Preferably both first- and second-strand cDNA synthesis is produced byreverse transcription, wherein DNA is made from RNA using the enzymereverse transcriptase. Reverse transcriptase is found in allretroviruses and is commonly obtained from avian myeloblastoma virus orMoloney murine leukemia virus; enzyme from these sources is commerciallyavailable from Life Technologies (Gaithersburg, Md.) and BoehringerMannheim (Indianapolis, Ind.).

The catalytic activities required to convert the promoter-primer-mRNAhybrid to double-stranded cDNA are a RNA-dependent DNA polymeraseactivity, a RNaseH activity, and a DNA-dependent DNA polymeraseactivity. Most reverse transcriptases, including those derived fromMoloney murine leukemia virus (MMLV-RT), avian myeloblastosis virus(AMV-RT), bovine leukemia virus (BLV-RT), Rous sarcoma virus (RSV) andhuman immunodeficiency virus (HIV-RT) catalyze each of these activities.These reverse transcriptases are sufficient to convert a primer-mRNAhybrid to double-stranded DNA in the presence of additional reagentsthat include, but are not limited to: dNTPs; monovalent and divalentcations, e.g., KCl, MgCl₂; sulfhydryl reagents, e.g., dithiothreitol;and buffering agents, e.g., Tris-Cl. Alternatively, a variety ofproteins that catalyze one or two of these activities can be added tothe cDNA synthesis reaction. For example, MMLV reverse transcriptaselacking RNaseH activity (described in U.S. Pat. No. 5,405,776) catalyzesRNA-dependent DNA polymerase activity and DNA-dependent DNA polymeraseactivity. These proteins may be added together during a single reactionstep, or added sequentially during two or more substeps. Preferably,MMLV is used for both first-and second-strand cDNA synthesis. Asdescribed above, preferably the reverse transcriptase is inactivatedprior to or inhibited during the transcription step of the method.

In general, it is preferable for the RNA-containing sample to containpurified poly-A⁺ RNA (mRNA). In one embodiment, a random promoter-primeris hybridized with an initial mRNA (poly-A⁺ RNA) sample. Thepromoter-primer is contacted with the mRNA under conditions that allowthe poly-dN site to hybridize to the mRNA. The randompromoter-primer-mRNA hybrid is then converted to a double-stranded cDNAproduct that is recognized by a RNA polymerase.

In a preferred embodiment, first-strand cDNA synthesis is allowed toproceed at a lower temperature (for example, 25° C.) for a certainperiod of time (e.g., 10 min) prior to increasing the temperature (e.g.,to 40° C.) for the remainder of the reverse transcription reaction,which improves annealing of the first primer (e.g., the promoter-primer)to its target nucleic acid sequence.

In the subject methods, conversion of the primer-mRNA hybrid todouble-stranded cDNA proceeds by priming second strand cDNA synthesiswith a random primer in the presence of a DNA-dependent DNA polymeraseactivity.

In another embodiment, the primer for second strand cDNA synthesis is amixture of primers consisting of a poly-dN sequence that is sufficientlylong to provide for efficient hybridization to the mRNA. The sequencetypically ranges in length from 4-50, preferably 6-25, more preferably6-12 or 6-9 and most preferably 6 degenerate bases, i.e., a randomhexamer (dN₆), wherein the degenerate bases may be A, T, G, or C. (Intheory, the primer should hybridize on average 4⁶ or 4096 base pairsfrom the 3′ priming site of the first-strand cDNA.) In specificembodiments, the poly-dN sequence is 4, 5, 6, 7, 8, 9, 10, 11, or 12nucleotides in length.

In a specific embodiment, the random primers used to prime second strandcDNA synthesis will be a complete set of all combinations of randomhexamers, i.e., a total of 4⁶ or 4096 hexamers.

Additional proteins that may enhance the yield of double-stranded DNAproducts may also be added to the cDNA synthesis reaction. Theseproteins include a variety of DNA polymerases (such as those derivedfrom E. coli, thermophilic bacteria, archaebacteria, phage, yeasts,Neurosporas, Drosophilas, primates and rodents), and DNA ligases (suchas those derived from phage or cellular sources, including T4 DNA ligaseand E. coli DNA ligase).

The second strand cDNA synthesis results in the creation of adouble-stranded promoter region. The second strand cDNA includes notonly a sequence of nucleotide residues that comprise a DNA copy of themRNA template, but also additional sequences at its 3′ end that arecomplementary to the promoter-primer used to prime first strand cDNAsynthesis. The double-stranded promoter region serves as a recognitionsite and transcription initiation site for RNA polymerase, which usesthe second strand cDNA as a template for multiple rounds of RNAsynthesis during the next stage of the subject methods (see Section 5.2,“Transcription of cDNA,” below).

Depending on the particular protocol, the same or different DNApolymerases may be employed during the cDNA synthesis step. In apreferred embodiment, a single reverse transcriptase, most preferablyMMLV-RT, is used as a source of all the requisite activities necessaryto convert the primer-mRNA hybrid to double-stranded cDNA. In anotherpreferred embodiment, the polymerase employed in first strand cDNAsynthesis is different from that which is employed in second strand cDNAsynthesis. Specifically, a reverse transcriptase lacking RNaseH activity(e.g., SUPERSCRIPT II™) is combined with the primer-mRNA hybrid during afirst substep for first strand synthesis. A source of RNaseH activity,such as E. coli RNaseH or MMLV-RT, but most preferably MMLV-RT, is addedduring a second substep to initiate second strand synthesis.

In yet other embodiments, the requisite activities are provided by aplurality of distinct enzymes. The manner in which double-stranded cDNAis produced from the initial mRNA is not critical to certain embodimentsof the invention. However, the preferred embodiments use MMLV-RT, or acombination of SUPERSCRIPT II™ and MMLV-RT, or a combination ofSUPERSCRIPT IITM and E. coli RNaseH, for cDNA synthesis as theseembodiments yield certain desired results. Specifically, in thepreferred embodiments, reaction conditions were chosen so that enzymespresent during the cDNA synthesis do not adversely affect the subsequenttranscription reaction. Potential inhibitors include, but are notlimited to, RNase contaminants of certain enzyme preparations.

5.2. Transcription of cDNA

The next step of the subject method is the preparation of RNA from thedouble-stranded cDNA prepared in the first step. During this step, thedouble-stranded cDNA produced in the first step is transcribed by RNApolymerase to yield RNA that, in certain embodiments, is complementaryto the initial nucleic acid target from which it is amplified. This stepis sometimes referred to as “in vitro transcription” (IVT).

The promoter regions that find use in the methods of the invention areregions where RNA polymerase binds tightly to the DNA and contain thestart site and signal for RNA synthesis to begin. A wide variety ofpromoters are known and many are very well characterized. In general,prokaryotic promoters are preferred over eukaryotic promoters, and phageor virus promoters most preferred. The RNA polymerase promoter sequenceis therefore preferably derived from a prokaryote such as E. coli or thebacteriophage T7, SP6, and T3, with the T7 RNA polymerase promotersequence particularly preferred. T7, T3 and SP6 promoter regions aredescribed in Chamberlin and Ryan, The Enzymes (ed. P. Boyer, AcademicPress, New York) (1982) pp 87-108, which excerpt is hereby incorporatedby reference in its entirety.

The RNA polymerase used for transcription must be capable of binding tothe particular RNA polymerase promoter sequence contained in the primer;hence usually the RNA polymerase promoter sequence and the polymerasewill be homologous. For example, if the T7 RNA polymerase promotersequence is employed in the primer, it is preferred to use T7 RNApolymerase to drive transcription. T7 polymerase is commerciallyavailable from several sources, including Promega Biotech (Madison,Wis.) and Epicenter Technologies (Madison, Wis.).

In a preferred embodiment, the random promoter-primer used to primefirst strand cDNA synthesis comprises a T7 promoter sequence-dN₉, andthe RNA polymerase employed is T7 RNA polymerase.

Preferably, the RNA polymerase promoter sequence is located at or nearthe terminus of the primer, in an orientation permitting transcriptionof the RNA population under study.

For this transcription step, the presence of the RNA polymerase promoterregion on the double-stranded cDNA is exploited for the production ofsense and/or antisense RNA. To synthesize the RNA, the double-strandedDNA is contacted with the appropriate RNA polymerase in the presence ofthe four ribonucleotides, under conditions sufficient for RNAtranscription to occur.

In one embodiment, the conditions for RNA transcription are thosedescribed in Section 6, Example 1. Briefly, the transcription mix andthe transcription reaction are as follows. 60 μl of Transcription Mixare aliquoted into each sample tube. The transcription reactions areincubated at 40° C. for 16 hrs.

Transcription Mix Component Volume (μl) Nuclease-free water 22.8 5xTranscription Buffer 16 100 mM DTT 6.0 NTPs (25 mM A, G, C, 6.0 mM UTP)8.0 as UTP (allylamine-derivatized UTP) 2.0 (75 mM) 200 mM MgCl₂ 3.3RNAGuard ™, Pharmacia (36 U/μl) 0.5 Inorganic Pyrophosphatase (200 U/ml)0.6 T7 RNA polymerase (2500 U/μl) 0.8 Volume of Transcription Mix 60

Composition of Transcription Reaction Final concentration Component oramount Double-strand cDNA Approximately 400 ng Tris-HCl, pH 7.5 52 mMMgCl₂ 15 mM KCl 19 mM NaCl 10 mM Spermidine 2 mM DTT 10 mM ATP, GTP, CTP2.5 mM each UTP 0.6 mM aa UTP 1.9 mM T7 RNA polymerase 2000 URNAGuard ™, Pharmacia 18 U Inorganic pyrophosphatase 0.12 U Totalreaction volume 80 μl

Other suitable conditions for RNA transcription using RNA polymerasesare known in the art, see e.g., Milligan and Uhlenbeck (1989), Methodsin Enzymol. 180, 51 (which is hereby incorporated by reference in itsentirety).

In one aspect of the invention, the transcription step is carried out inthe presence of reverse transcriptase that is present in the reactionmixture from the double-stranded cDNA synthesis. Thus, the subjectmethods do not involve a step in which the double-stranded cDNA isphysically separated from the reverse transcriptase followingdouble-stranded cDNA preparation facilitating high throughputamplification and analysis. In this aspect of the invention, the reversetranscriptase that is present during the transcription step is renderedinactive. Thus, the transcription step is carried out in the presence ofa reverse transcriptase that is unable to catalyze RNA-dependent DNApolymerase activity, at least for the duration of the transcriptionstep. As a result., the RNA products of the transcription reactioncannot serve as substrates for additional rounds of cDNA synthesis, andthe amplification process cannot proceed exponentially.

The reverse transcriptase present during the transcription step may berendered inactive using any convenient protocol. The transcriptase maybe irreversibly or reversibly rendered inactive. Where the transcriptaseis reversibly rendered inactive, the transcriptase is physically orchemically altered so as to no longer be able to catalyze RNA-dependentDNA polymerase activity. The transcriptase may be irreversiblyinactivated by any convenient means. Thus, the reverse transcriptase maybe heat inactivated, in which the reaction mixture is subjected toheating to a temperature sufficient to inactivate the reversetranscriptase prior to commencement of the transcription step. In theseembodiments, the temperature of the reaction mixture and therefore thereverse transcriptase present therein is typically raised to 55° C. to70° C. for 5 to 60 minutes, usually to about 65° C. for 15 to 20minutes.

Alternatively, reverse transcriptase may be irreversibly inactivated byintroducing a reagent into the reaction mixture that chemically altersthe enzyme so that it no longer has RNA-dependent DNA polymeraseactivity. In yet other embodiments, the reverse transcriptase isreversibly inactivated. In these embodiments, the transcription step maybe carried out in the presence of an inhibitor of RNA-dependent DNApolymerase activity. Any convenient reverse transcriptase inhibitor maybe employed that is capable of inhibiting RNA-dependent DNA polymeraseactivity a sufficient amount to provide for linear amplification.However, these inhibitors should not adversely affect RNA polymeraseactivity. Reverse transcriptase inhibitors of interest include ddNTPs,such as ddATP, ddCTP, ddGTP or ddTTP, or a combination thereof, thetotal concentration of the inhibitor typically ranges from about 50 μMto 200 μM.

Because of the nature of the subject methods, all of the necessarypolymerization reactions, i.e., first strand cDNA synthesis, secondstrand cDNA synthesis and RNA transcription, may be carried out in thesame reaction vessel at the same temperature, such that temperaturecycling is not required. As such, the subject methods are particularlysuited for automation, as the requisite reagents for each of the abovesteps need merely be added to the reaction mixture in the reactionvessel, without any complicated separation steps being performed, suchas phenol/chloroform extraction. A further feature of the subjectinvention is that, despite its simplicity, it yields high amplificationextents, where the amplification extents (mass of RNA product/mass ofnucleic acid target) typically are at least about 50-fold, usually atleast about 200-fold and may be as high as 600-fold or higher.Furthermore, such amplification extents are achieved with lowvariability, e.g., coefficients of variation about the meanamplification extents that do not exceed about 10%, and usually do notexceed about 5%.

The resultant cRNA (particularly antisense RNA) produced by the subjectmethods finds use in a variety of applications. RNA amplified by themethods of the invention may be labeled and employed to profile geneexpression in different populations of cells. In a preferred embodiment,the amplified RNA is used for quantitative comparisons of geneexpression between different populations of cells or between populationsof cells exposed to different stimuli. For example, the resultantantisense RNA can be used in expression profiling analysis on suchplatforms as DNA microarrays, for construction of “driver” forsubtractive hybridization assays, for cDNA library construction, and thelike. Especially facilitated by the subject methods are studies ofdifferential gene expression in mammalian cells or cell populations. Thecells may be from blood (e.g., white cells, such as T or B cells) orfrom tissue derived from solid organs, such as brain, spleen, bone,heart, vascular, lung, kidney, liver, pituitary, endocrine glands, lymphnode, dispersed primary cells, tumor cells, or the like.

The RNA amplification technology can also be applied to improve methodsof detecting and isolating nucleic acid sequences that vary in abundanceamong different populations using the technique known as subtractivehybridization. In such assays, two nucleic acid populations, one senseand the other antisense, are allowed to mix with one another with onepopulation being present in molar excess (“driver”). Under appropriateconditions, the sequences represented in both populations form hybrids,whereas sequences present in only one population remain single-stranded.Thereafter, various well known techniques are used to separate theunhybridized molecules representing differentially expressed sequences.The amplification technology described herein may be used to constructlarge amounts of antisense RNA for use as “driver” in such experiments.

5.3. Oligonucleotides

A primer may be prepared by any suitable method, such as phosphotriesterand phosphodiester methods of synthesis, or automated embodimentsthereof. It is also possible to use a primer that has been isolated froma biological source, such as a restriction endonuclease digest, althougha synthetic primer is preferred.

An oligonucleotide primer can be DNA, RNA, chimeric mixtures orderivatives or modified versions thereof, so long as it is still capableof priming the desired reaction. The oligonucleotide primer can bemodified at the base moiety, sugar moiety, or phosphate backbone, andmay include other appending groups or labels, so long as it is stillcapable of priming the desired amplification reaction.

For example, an oligonucleotide primer may comprise at least onemodified base moiety which is selected from the group including but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. In another embodiment, the oligonucleotide primercomprises at least one modified sugar moiety selected from the groupincluding but not limited to arabinose, 2-fluoroarabinose, xylulose, andhexose.

In yet another embodiment, the oligonucleotide primer comprises at leastone modified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

An oligonucleotide primer for use in the methods of the invention may bederived by cleavage of a larger nucleic acid fragment using non-specificnucleic acid cleaving chemicals or enzymes or site-specific restrictionendonucleases; or by synthesis by standard methods known in the art,e.g., by use of an automated DNA synthesizer (such as are commerciallyavailable from Biosearch, Applied Biosystems, etc.) and standardphosphoramidite chemistry. As examples, phosphorothioateoligonucleotides may be synthesized by the method of Stein et al. (1988,Nucl. Acids Res. 16:3209-3221), methylphosphonate oligonucleotides canbe prepared by use of controlled pore glass polymer supports (Sarin etal., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.

Once the desired oligonucleotide is synthesized, it is cleaved from thesolid support on which it was synthesized and treated, by methods knownin the art, to remove any protecting groups present. The oligonucleotidemay then be purified by any method known in the art, includingextraction and gel purification. The concentration and purity of theoligonucleotide may be determined by examining oligonucleotide that hasbeen separated on an acrylamide gel, or by measuring the optical densityat 260 nm in a spectrophotometer.

5.4. Methods of Labeling of Nucleic Acid Amplification Products

Nucleic acid amplification products such as amplified RNA may be labeledwith any art-known detectable marker, including radioactive labels suchas ³²P, ³⁵S, ³H, and the like; fluorophores; chemiluminescers; orenzymatic markers. In a preferred embodiment, the label is fluorescent.Exemplary suitable fluorophore moieties that can be selected as labelsare listed in Table 1.

TABLE 1 Suitable fluorophore moieties that can be selected as labels4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid acridine andderivatives: acridine acridine isothiocyanate5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS)4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (LuciferYellow VS)-(4-anilino-1-naphthyl)maleimide anthranilamide BrilliantYellow coumarin and derivatives: coumarin 7-amino-4-methylcoumarin (AMC,Coumarin 120) 7-amino-4-trifluoromethylcoumarin (Coumarin 151) Cy3 Cy5cyanosine 4′,6-diaminidino-2-phenylindole (DAPI)5′,5″-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red)7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl chloride)4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL)4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC) eosin andderivatives: eosin eosin isothiocyanate erythrosin and derivatives:erythrosin B erythrosin isothiocyanate ethidium fluorescein andderivatives: 5-carboxyfluorescein (FAM)5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF)2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE) fluoresceinfluorescein isothiocyanate QFITC (XRITC) fluorescamine IR 144 IR1446Malachite Green isothiocyanate 4-methylumbelliferone orthocresolphthalein nitrotyrosine pararosaniline Phenol Red B-phycoerythrino-phthaldialdehyde pyrene and derivatives: pyrene pyrene butyratesuccinimidyl 1-pyrene butyrate Reactive Red 4 (Cibacron ® Brilliant Red3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX)6-carboxyrhodamine (R6G) lissamine rhodamine B sulfonyl chloriderhodamine (Rhod) rhodamine B rhodamine 110 rhodamine 123 rhodamine Xisothiocyanate sulforhodamine B sulforhodamine 101 sulfonyl chloridederivative of sulforhodamine 101 (Texas Red)N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodaminetetramethyl rhodamine isothiocyanate (TRITC) riboflavin rosolic acidterbium chelate derivatives

5.4.1. Labeling of RNA

Depending on the particular intended use of the RNA amplificationproducts, the RNA amplification products may be labeled. The RNA may belabeled with any art-known detectable marker, including but not limitedto radioactive labels such as ³²P, ³⁵S, ³H, and the like; fluorophores;chemiluminescers; or enzymatic markers (e.g., as listed in Table 1).

Labeling of RNA is preferably accomplished by including one or morelabeled NTPs in the in vitro transcription (IVT) reaction mixture. NTPsmay be directly labeled with a radioisotope, such as ³²P, ³⁵S, ³H;radiolabeled NTPs are available from several sources, including NewEngland Nuclear (Boston, Mass.) and Amersham. NTPs may be directlylabeled with a fluorescent label such as Cy3 or Cy5. In one embodiment,biotinylated or allylamine-derivatized NTPs are incorporated during theIVT reaction and the resultant cRNAs are thereafter labeled, forexample, by the addition of fluorophore-conjugated avidin, in the caseof biotin, or the NHS ester of a fluorophore, in the case of allylamine.In another embodiment, fluorescently labeled NTPs may be incorporatedduring the IVT reaction, which fluorescently labels the resultant cRNAsdirectly.

RNA may be fluorescently labeled with fluorescently tagged nucleotides(e.g., fluorescently labeled ATP, UTP, GTP or CTP) that are incorporatedinto the antisense RNA product during the transcription step.Fluorescent moieties that may be used to tag nucleotides for producinglabeled antisense RNA include: fluorescein, the cyanine dyes, such asCy3, Cy5, Alexa 542, Bodipy 630/650, and the like. Other labels may alsobe employed as are known in the art. Exemplary fluorophore moieties thatcan be used as labels are listed in Table 1. The preferred label in thesubject methods is a fluorophore, such as fluorescein isothiocyanate,lissamine, Cy3, Cy5, and rhodamine 110, with Cy3 and Cy5 particularlypreferred.

Not only fluorophores, but also chemiluminescers and enzymes, amongothers, may be used as labels. In yet another embodiment, the RNA islabeled with an enzymatic marker that produces a detectable signal whena particular chemical reaction is conducted, such as alkalinephosphatase or horseradish peroxidase. Such enzymatic markers arepreferably heat stable, so as to survive the denaturing steps of theamplification process.

RNA may also be indirectly labeled by incorporating a nucleotide linkedcovalently to a hapten or to a molecule such as biotin, to which alabeled avidin molecule may be bound, or digoxygenin, to which a labeledanti-digoxygenin antibody may be bound. RNA may be labeled with labelingmoieties during chemical synthesis or the label may be attached aftersynthesis by methods known in the art.

Labeling of RNA is preferably accomplished by preparing cRNA that isfluorescently labeled with NHS-esters. Most preferably, labeling of RNAis accomplished in a two-step procedure in which allylamine-derivatizedUTP (aa UTP) is incorporated during IVT. Following the IVT reaction,unincorporated nucleotides are removed and the allylamine-containingRNAs are conjugated to the N-hydroxysuccinimide (NHS) esters of Cy3 orCy5.

In a preferred embodiment, 5-(3-Aminoallyl)uridine 5′-triphosphate isincorporated into the RNA amplification product during transcription andpost-synthetically coupled to Cy-NHS, either Cy3-NHS or Cy5-NHS.

In a specific embodiment, a two-step method of preparingfluorescent-labeled cRNA may be used in two color hybridizations to DNAmicroarrays. Such a two-step method is disclosed in U.S. Ser. No.09/411,074, filed Oct. 4, 1999, the disclosure of which is hereinincorporated by reference. In one embodiment, aminoallyl (aa)-labelednucleic acids are prepared by incorporation of aa-nucleotides. aa-UTP(Sigma A-5660) may be used for labeling cRNA. aa-cRNA is prepared usingthe Ambion MegaScript T7 RNA polymerase in vitro transcription kit, withaa-UTP substituted at 50-100% of the total UTP concentration. It isessential to remove all traces of amine-containing buffers such as Trisprior to derivatizing the aa-nucleic acids. aa-Nucleic acids prepared inenzymatic reactions are preferably cleaned up on appropriate QIAGENcolumns: RNeasy® Mini kit (for RNA) or QIAquick PCR Purification kit(for DNA) (QIAGEN Inc.—USA, Valencia, Calif.). For the QIAGEN columns,samples are applied twice. For washes, 80% EtOH is preferablysubstituted for the buffer provided with the QIAGEN kit. Samples areeluted twice with 50 μl volumes of 70° C. H₂O. Alternatively (but lesspreferably), samples may be cleaned up by repeated cycles of dilutionand concentration on Microcon-30 filters.

In a second step of the embodiment, aa-nucleic acids are derivatizedwith NHS-esters, preferably Cy 3 or Cy 5. Preferably, 2-6 μg ofaa-labeled nucleic acid are aliquoted into a microfuge tube, adjustingthe total volume to 12 μl with H₂O. The NHS-ester is dissolved at aconcentration of ˜15 mM in anhydrous DMSO (˜200 nmoles in 13 μl). 27 μlof 0.1 M sodium carbonate buffer, pH 9, are added. 12 μl of the dye mix(containing ˜60 nmoles dye-NHS ester) are then immediately added to theaa-labeled nucleic acid (˜6-20 pmoles of a 1 kb molecule). The samplesare then incubated in the dark at 23° C. for 1 hour. The couplingreaction is stopped by adding 5 μl of a 4M solution of hydroxylamine.Incubation is continued at 23° C. for an additional 0.25 hr. Dye-couplednucleic acid is separated from unincorporated dye on an RNeasy® Mini kitor QIAquick PCR Purification kit (QIAGEN Inc.—USA, Valencia, Calif.).Samples are washed with 80% EtOH instead of buffer, as described above,and eluted twice with 50 μl volumes of 70° C. H₂O.

The spectrum of the labeled nucleic acid is preferably measured from 220nm-700 nm. The percent recovery of nucleic acid and molar incorporationof dye is calculated from extinction coefficients and absorbance valuesat 1_(max). Recovery of nucleic acid is typically ˜80%. The mole percentof dye incorporated per nucleotide ranges from 1.5-5% of totalnucleotides.

Often it is desired to compare gene expression in two differentpopulations of cells, perhaps derived from different tissues or perhapsexposed to different stimuli. Such comparisons are facilitated bylabeling the RNAs from one population with a first fluorophore and theRNAs from the other population, with a second fluorophore, where the twofluorophores have distinct emission spectra. Again, Cy3 and Cy5 areparticularly preferred fluorophores for use in comparing gene expressionbetween two different populations of cells.

5.5. Methods of Preparation of Source RNA

The source RNA may he obtained from a variety of different sources,typically a biological source. In specific embodiments, the biologicalsource may be any of a variety of eukaryotic sources. Biological sourcesof interest may include sources derived from single-celled organismssuch as yeast and multicellular organisms, including plants and animals,particularly mammals. Biological sources from multicellular organismsmay be derived from particular organs or tissues of the multicellularorganism, or from isolated cells derived therefrom. In obtaining thesample of RNA to be analyzed from its biological source, the source maybe subjected to a number of different processing steps. Such processingsteps might include tissue homogenization, cell isolation followed bycytoplasm extraction or isolation, nucleic acid extraction and the like.Such processing steps for isolating RNA from its biological source areknown to those of skill in the art. For example, methods of isolatingRNA from cells, tissues, organs or whole organisms are described inSambrook et al. (1989), Molecular Cloning: A Laboratory Manual 2d Ed.(Cold Spring Harbor Press), incorporated herein by reference in itsentirety. Alternatively, at least some of the initial steps of thesubject methods may be performed in situ, as described in Eberwine (U.S.Pat. No. 5,514,545, entitled “Method for characterizing single cellsbased on RNA amplification for diagnostics and therapeutics,” issued May7, 1996), the disclosure of which is herein incorporated by reference.

Although the amplification methods of the invention can be adapted toamplify DNA, it is preferred to utilize the methods to amplify RNA froma population of cells. Total cellular RNA, cytoplasmic RNA, or poly(A)⁺RNA may be used, with poly(A)⁺ RNA (mRNA) being preferred. Methods forpreparing total and poly(A)⁺ RNA are well known and are describedgenerally in Sambrook et al. (1989, Molecular Cloning—A LaboratoryManual (2nd Ed), Vols. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.) and Ausubel et al., eds. (1994, Current Protocols inMolecular Biology, vol. 2, Current Protocols Publishing, New York),incorporated herein by reference in their entireties.

RNA may be isolated from eukaryotic cells by procedures that involvelysis of the cells and denaturation of the proteins contained therein.Cells of interest include wild-type cells, drug-exposed wild-type cells,modified cells, and drug-exposed modified cells.

Additional steps may be employed to remove DNA. Cell lysis may beaccomplished with a nonionic detergent, followed by microcentrifugationto remove the nuclei and hence the bulk of the cellular DNA. In oneembodiment, RNA is extracted from cells of the various types of interestusing guanidinium thiocyanate lysis followed by CsCl centrifugation toseparate the RNA from DNA (Chirgwin et al., 1979, Biochemistry18:5294-5299). Poly(A)⁺ RNA is selected by selection with oligo-dTcellulose (see Sambrook et al., 1989, Molecular Cloning—A LaboratoryManual (2nd Ed), Vols. 1-3, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). Alternatively, separation of RNA from DNA can beaccomplished by organic extraction, for example, with hot phenol orphenol/chloroform/isoamyl alcohol.

If desired, RNase inhibitors may be added to the lysis buffer. Likewise,for certain cell types, it may be desirable to add a proteindenaturation/digestion step to the protocol.

For many applications, it is desirable to preferentially enrich mRNAwith respect to other cellular RNAs, such as transfer RNA (tRNA) andribosomal RNA (rRNA). Most mRNAs contain a poly(A) tail at their 3′ end.This allows them to be enriched by affinity chromatography, for example,using oligo(dT) or poly(U) coupled to a solid support, such as celluloseor Sephadex™ (see Ausubel et al., eds., 1994, Current Protocols inMolecular Biology, vol. 2, Current Protocols Publishing, New York). Oncebound, poly(A)+[+?] mRNA is eluted from the affinity column using 2 mMEDTA/0.1% SDS.

The sample of RNA can comprise a plurality of different mRNA molecules,each different mRNA molecule having a different nucleotide sequence. Ina specific embodiment, the mRNA molecules in the RNA sample comprise atleast 100 different nucleotide sequences. More preferably, the mRNAmolecules of the RNA sample comprise at least 500, 1,000, 5,000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,[?] 90,000 or100,000 different nucleotide sequences. In another specific embodiment,the RNA sample is a mammalian RNA sample, the mRNA molecules of themammalian RNA sample comprising about 20,000 to 30,000 differentnucleotide sequences.

In a specific embodiment, total RNA or mRNA from cells are used in themethods of the invention. The source of the RNA can be cells of a plantor animal, human, mammal, primate, non-human animal, dog, cat, mouse,rat, rabbit, bird, yeast, eukaryote, prokaryote, etc. In one embodiment,the method of the invention is used with a sample containing total mRNAor total RNA from 1×10⁶ cells or less.

5.6. Methods for Determining Biological Response Profiles

This section provides some exemplary methods for measuring biologicalresponses using cRNA amplified by methods of the invention. One of skillin the art would appreciate that this invention is not limited to thefollowing specific methods for measuring the responses of a biologicalsystem, i.e., gene expression profiles. In particular, the presence ofcRNA(s) of interest (and thus mRNA(s) of interest in the sample) can bedetected or measured by procedures including, but not limited to,Northern blotting, the use of oligonucleotides tethered to beads asprobes, or the use of polynucleotide microarrays.

In a specific embodiment of the invention, one or more labels isintroduced into the RNA during the transcription step to facilitate geneexpression profiling. Gene expression can be profiled in any of severalways, among which the preferred method is to probe a DNA microarray withthe labeled RNA transcripts generated above. A DNA microarray, or chip,is a microscopic array of DNA fragments or synthetic oligonucleotides,disposed in a defined pattern on a solid support, wherein they areamenable to analysis by standard hybridization methods (Schena,BioEssays 18: 427, 1996).

The DNA in a microarray may be derived from genomic or cDNA libraries,from fully sequenced clones, or from partially sequenced cDNAs known asexpressed sequence tags (ESTs). Methods for obtaining such DNA moleculesare generally known in the art (see, e.g., Ausubel et al., eds., 1994,Current Protocols in Molecular Biology, vol. 2, Current ProtocolsPublishing, New York). Alternatively, oligonucleotides may besynthesized by conventional methods, such as phosphoramidite-basedsynthesis.

Gene expression profiling can be done for purposes of screening,diagnosis, staging a disease, and monitoring response to therapy, aswell as for identifying genetic targets of drugs and of pathogens.

5.6.1. Transcript Assay Using DNA Arrays

This invention is particularly useful for the analysis of geneexpression profiles. For expression profiling, DNA microarrays aretypically probed using mRNA, extracted and amplified from the cellswhose gene expression profile it is desired to analyze, using therandom-primed RT-IVT amplification method of the invention. Tofacilitate comparison between any two samples of interest, thepolynucleotides representing the mRNA transcripts present in a cell aretypically labeled separately with fluorescent dyes that emit atdifferent wavelengths. Some embodiments of this invention are based onmeasuring the transcriptional rate of genes.

The transcriptional rate can be measured by techniques of hybridizationto arrays of nucleic acid or nucleic acid mimic probes, described in thenext subsection, or by other gene expression technologies, such as thosedescribed in the subsequent subsection. However measured, the result iseither the absolute, relative amounts of transcripts or response dataincluding values representing RNA abundance ratios, which usuallyreflect DNA expression ratios (in the absence of differences in RNAdegradation rates).

In various alternative embodiments of the present invention, aspects ofthe biological state other than the transcriptional state, such as thetranslational state, the activity state, or mixed aspects can bemeasured.

Preferably, measurement of the transcriptional state is made byhybridization to transcript arrays, which are described in thissubsection. Certain other methods of transcriptional state measurementare described later in this subsection. In a preferred embodiment thepresent invention makes use of “transcript arrays” (also called herein“microarrays”). Transcript arrays can be employed for analyzing thetranscriptional state in a biological sample and especially formeasuring the transcriptional states of a biological sample exposed tograded levels of a drug of interest or to graded perturbations to abiological pathway of interest.

In one embodiment, transcript arrays are produced by hybridizingdetectably labeled polynucleotides representing the mRNA transcriptspresent in a cell (e.g., fluorescently labeled cRNA that is amplified bythe methods of the present invention) to a microarray. A microarray is asurface with an ordered array of binding (e.g., hybridization) sites forproducts of many of the genes in the genome of a cell or organism,preferably most or almost all of the genes. Microarrays can be made in anumber of ways, of which several are described below. However produced,microarrays share certain preferred characteristics: The arrays arereproducible, allowing multiple copies of a given array to be producedand easily compared with each other. Preferably the microarrays aresmall, usually smaller than 5 cm², and they are made from materials thatare stable under binding (e.g., nucleic acid hybridization) conditions.A given binding site or unique set of binding sites in the microarraywill specifically bind the product of a single gene in the cell.Although there may be more than one physical binding site (hereinafter“site”) per specific mRNA, for the sake of clarity the discussion belowwill assume that there is a single site.

In one embodiment, the microarray is an array of polynucleotide probes,the array comprising a support with at least one surface and at least100 different polynucleotide probes, each different polynucleotide probecomprising a different nucleotide sequence and being attached to thesurface of the support in a different location on the surface.Preferably, the nucleotide sequence of each of the differentpolynucleotide probes is in the range of 40 to 80 nucleotides in length.More preferably, the nucleotide sequence of each of the differentpolynucleotide probes is in the range of 50 to 70 nucleotides in length.Even more preferably, the nucleotide sequence of each of the differentpolynucleotide probes is in the range of 50 to 60 nucleotides in length.

In specific embodiments, the array comprises polynucleotide probes of atleast 2,000, 4,000, 10,000, 15,000, 20,000, 50,000, 80,000, or 100,000different nucleotide sequences.

In another embodiment, the nucleotide sequence of each polynucleotideprobe in the array is specific for a particular target polynucleotidesequence. In yet another embodiment, the target polynucleotide sequencescomprise expressed polynucleotide sequences of a cell or organism.

In a specific embodiment, the cell or organism is a mammalian cell ororganism. In another specific embodiment, the cell or organism is ahuman cell or organism. In specific embodiments, the nucleotidesequences of the different polynucleotide probes of the array arespecific for at least 50%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% of the genes in the genome ofthe cell or organism. Most preferably, the nucleotide sequences of thedifferent polynucleotide probes of the array are specific for all of thegenes in the genome of the cell or organism.

In specific embodiments, the polynucleotide probes of the arrayhybridize specifically and distinguishably to at least 10,000, to atleast 20,000, to at least 50,000, different polynucleotide sequences, toat least 80,000, or to at least 100,000 different polynucleotidesequences.

In other specific embodiments, the polynucleotide probes of the arrayhybridize specifically and distinguishably to at least 90%, at least95%, or at least 99% of the genes or gene transcripts of the genome of acell or organism. Most preferably, the polynucleotide probes of thearray hybridize specifically and distinguishably to the genes or genetranscripts of the entire genome of a cell or organism.

In specific embodiments, the array has at least 100, at least 250, atleast 1,000, or at least 2,500 probes per 1 cm², preferably all or atleast 25% or 50% of which are different from each other.

In another embodiment, the array is a positionally addressable array (inthat the sequence of the polynucleotide probe at each position isknown).

In another embodiment, the nucleotide sequence of each polynucleotideprobe in the array is a DNA sequence. In another embodiment, the DNAsequence is a single-stranded DNA sequence. The DNA sequence may be,e.g., a cDNA sequence, or a synthetic sequence.

When cRNA complementary to the RNA of a cell is made and hybridized to amicroarray under suitable hybridization conditions, the level ofhybridization to the site in the array corresponding to any particulargene will reflect the prevalence in the cell of mRNA transcribed fromthat gene. For example, when detectably labeled (e.g., with afluorophore) cRNA complementary to the total cellular mRNA is hybridizedto a microarray, the site on the array corresponding to a gene (i.e.,capable of specifically binding the product of the gene) that is nottranscribed in the cell will have little or no signal (e.g., fluorescentsignal), and a gene for which the encoded mRNA is prevalent will have arelatively strong signal.

In preferred embodiments, cRNAs from two different cells are hybridizedto the binding sites of the microarray. In the case of drug responsesone biological sample is exposed to a drug and another biological sampleof the same type is not exposed to the drug. In the case of pathwayresponses one cell is exposed to a pathway perturbation and another cellof the same type is not exposed to the pathway perturbation. The cRNAderived from each of the two cell types are differently labeled so thatthey can be distinguished. In one embodiment, for example, cRNA from acell treated with a drug (or exposed to a pathway perturbation) issynthesized using a fluorescein-labeled NTP, and cRNA from a secondcell, not drug-exposed, is synthesized using a rhodamine-labeled NTP.When the two cRNAs are mixed and hybridized to the microarray, therelative intensity of signal from each cRNA set is determined for eachsite on the array, and any relative difference in abundance of aparticular mRNA detected.

In the example described above, the cRNA from the drug-treated (orpathway perturbed) cell will fluoresce green when the fluorophore isstimulated and the cRNA from the untreated cell will fluoresce red. As aresult, when the drug treatment has no effect, either directly orindirectly, on the relative abundance of a particular mRNA in a cell,the mRNA will be equally prevalent in both cells and, upon reversetranscription, red-labeled and green-labeled cRNA will be equallyprevalent. When hybridized to the microarray, the binding site(s) forthat species of RNA will emit wavelengths characteristic of bothfluorophores (and appear brown in combination). In contrast, when thedrug-exposed cell is treated with a drug that, directly or indirectly,increases the prevalence of the mRNA in the cell, the ratio of green tored fluorescence will increase. When the drug decreases the mRNAprevalence, the ratio will decrease.

The use of a two-color fluorescence labeling and detection scheme todefine alterations in gene expression has been described, e.g., inSchena et al., 1995, Science 270:467-470, which is incorporated byreference in its entirety for all purposes. An advantage of using eRNAlabeled with two different fluorophores is that a direct and internallycontrolled comparison of the mRNA levels corresponding to each arrayedgene in two cell states can be made, and variations due to minordifferences in experimental conditions (e.g., hybridization conditions)will not affect subsequent analyses. However, it will be recognized thatit is also possible to use cRNA from a single cell, and compare, forexample, the absolute amount of a particular mRNA in, e.g., adrug-treated or pathway-perturbed cell and an untreated cell.

5.6.2. Preparation of Microarrays

Microarrays are known in the art and consist of a surface to whichprobes that correspond in sequence to gene products (e.g., cDNAs, mRNAs,cRNAs, polypeptides, and fragments thereof), can be specificallyhybridized or bound at a known position. In one embodiment, themicroarray is an array (i.e., a matrix) in which each positionrepresents a discrete binding site for a product encoded by a gene(e.g., a protein or RNA), and in which binding sites are present forproducts of most or almost all of the genes in the organism's genome. Ina preferred embodiment, the “binding site” (hereinafter, “site”) is anucleic acid or nucleic acid analogue to which a particular cognate cRNAcan specifically hybridize. The nucleic acid or analogue of the bindingsite can be, e.g., a synthetic oligomer, a full-length cRNA, a less-thanfull length cRNA, or a gene fragment.

In one embodiment, the microarray contains binding sites for products ofall or almost all genes in the target organism's genome. This microarraywill have binding sites corresponding to at least about 50% of the genesin the genome, often at least about 75%, more often at least about 85%,even more often more than about 90%, and most often at least about 99%.

Such comprehensiveness, however, is not necessarily required. In anotherembodiment, the microarray contains binding sites for products of humangenes. This microarray will have binding sites corresponding to at leastabout 5-10% of the genes in the genome, preferably at least about10-15%, and more preferably at least about 40%.

Preferably, the microarray has binding sites for genes relevant to theaction of a drug of interest or in a biological pathway of interest. A“gene” is identified as an open reading frame (ORF) of preferably atleast 50, 75, or 99 amino acids from which a messenger RNA istranscribed in the organism (e.g., if a single cell) or in some cell ina multicellular organism. The number of genes in a genome can beestimated from the number of mRNAs expressed by the organism, or byextrapolation from a well-characterized portion of the genome. When thegenome of the organism of interest has been sequenced, the number ofORFs can be determined and mRNA coding regions identified by analysis ofthe DNA sequence. For example, the Saccharomyces cerevisiae genome hasbeen completely sequenced and is reported to have approximately 6275open reading frames (ORFs) longer than 99 amino acids. Analysis of theseORFs indicates that there are 5885 ORFs that are likely to specifyprotein products (Goffeau et al., 1996, Science 274:546-567, which isincorporated by reference in its entirety for all purposes). Incontrast, the human genome is estimated to contain approximately 10⁵genes.

5.6.3. Preparation of Nucleic Acids for Microarrays

As noted above, the “binding site” to which a particular cognate cRNAspecifically hybridizes is usually a nucleic acid or nucleic acidanalogue attached at that binding site. In one embodiment, the bindingsites of the microarray are DNA polynucleotides corresponding to atleast a portion of each gene in an organism's genome. These DNAs can beobtained by, e.g., polymerase chain reaction (PCR) amplification of genesegments from genomic DNA, cDNA (e.g., by reverse transcription orRT-PCR), or cloned sequences. Nucleic acid amplification primers arechosen, based on the known sequence of the genes or cDNA, that result inamplification of unique fragments (i.e., fragments that do not sharemore than 10 bases of contiguous identical sequence with any otherfragment on the microarray). Computer programs are useful in the designof primers with the required specificity and optimal amplificationproperties. See, e.g., Oligo version 5.0 (National Biosciences). In thecase of binding sites corresponding to very long genes, it willsometimes be desirable to amplify segments near the 3′ end of the geneso that when oligo-dT primed cDNA probes are hybridized to themicroarray, less-than-full length probes will bind efficiently.Typically each gene fragment on the microarray will be between about 50by and about 2000 bp, more typically between about 100 by and about 1000bp, and usually between about 300 by and about 800 by in length.

Nucleic acid amplification methods are well known and are described, forexample, in Innis et al., eds., 1990, PCR Protocols: A Guide to Methodsand Applications, Academic Press Inc., San Diego, Calif., which isincorporated by reference in its entirety for all purposes. It will beapparent that computer controlled robotic systems are useful forisolating and amplifying nucleic acids.

An alternative means for generating the nucleic acid for the microarrayis by synthesis of synthetic polynucleotides or oligonucleotides, e.g.,using N-phosphonate or phosphoramidite chemistries (e.g., Froehler etal., 1986, Nucleic Acid Res 14:5399-5407). Synthetic sequences arebetween about 15 and about 100 bases in length, preferably between about20 and about 50 bases.

In some embodiments, synthetic nucleic acids include non-natural bases,e.g., inosine. Where the particular base in a given sequence is unknownor is polymorphic, a universal base, such as inosine or 5-nitroindole,may be substituted. Additionally, it is possible to vary the charge onthe phosphate backbone of the oligonucleotide, for example, bythiolation or methylation, or even to use a peptide rather than aphosphate backbone. The making of such modifications is within the skillof one trained in the art.

As noted above, nucleic acid analogues may be used as binding sites forhybridization. An example of a suitable nucleic acid analogue is peptidenucleic acid (see, e.g., Egholm et al., 1993, Nature 365:566-568; seealso U.S. Pat. No. 5,539,083, Cook et al., entitled “Peptide nucleicacid combinatorial libraries and improved methods of synthesis,” issuedJul. 23, 1996).

In an alternative embodiment, the binding (hybridization) sites are madefrom plasmid or phage clones of genes, cDNAs (e.g., expressed sequencetags), or inserts therefrom (Nguyen et al., 1995, Genomics 29:207-209).In yet another embodiment, the polynucleotide of the binding sites isRNA.

5.6.4. Attaching Nucleic Acids to the Solid Surface

The nucleic acid or analogue are attached to a solid support, which maybe made from glass, silicon, plastic (e.g., polypropylene, nylon,polyester), polyacrylamide, nitrocellulose, cellulose acetate or othermaterials. In general, non-porous supports, and glass in particular, arepreferred. The solid support may also be treated in such a way as toenhance binding of oligonucleotides thereto, or to reduce non-specificbinding of unwanted substances thereto. Preferably, the glass support istreated with polylysine or silane to facilitate attachment ofoligonucleotides to the slide.

Methods of immobilizing DNA on the solid support may include directtouch, micropipetting (Yershov et al., Proc. Natl. Acad. Sci. USA (1996)93(10):4913-4918), or the use of controlled electric fields to direct agiven oligonucleotide to a specific spot in tine array (U.S. Pat. No.5,605,662, Heller et al., entitled “Active programmable electronicdevices for molecular biological analysis and diagnostics,” issued Feb.25, 1997). DNA is typically immobilized at a density of 100 to 10,000oligonucleotides per cm² and preferably at a density of about 1000oligonucleotides per cm².

A preferred method for attaching the nucleic acids to a surface is byprinting on glass plates, as is described generally by Schena et al.,1995, Science 270:467-470. This method is especially useful forpreparing microarrays of cDNA. (See also DeRisi et al., 1996, NatureGenetics 14:457-460; Shalon et al., 1996, Genome Res. 6:639-645; andSchena et al., Proc. Natl. Acad. Sci. USA, 1996, 93(20):10614-19.)

In a preferred alternative to immobilizing pre-fabricatedoligonucleotides onto a solid support, it is possible to synthesizeoligonucleotides directly on the support (Maskos et al., Nucl. AcidsRes. 21: 2269-70, 1993; Fodor et al., Science 251: 767-73, 1991;Lipshutz et al., 1999, Nat. Genet. 21(1 Suppl):20-4). Among methods ofsynthesizing oligonucleotides directly on a solid support, particularlypreferred methods are photolithography (see Fodor et al., Science 251:767-73, 1991; McGall et al., Proc. Natl. Acad Sci. (USA) 93: 13555-60,1996) and piezoelectric printing (Lipshutz et al., 1999, Nat. Genet.21(1 Suppl):20-4), with the piezoelectric method most preferred.

In one embodiment, a high-density oligonucleotide array is employed.Techniques are known for producing arrays containing thousands ofoligonucleotides complementary to defined sequences, at definedlocations on a surface using photolithographic techniques for synthesisin situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al.,1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; Lockhart et al., 1996,Nature Biotechnol. 14:1675-80; U.S. Pat. No. 5,578,832, Trulson et al.,entitled “Method and apparatus for imaging a sample on a device,” issuedNov. 26, 1996; U.S. Pat. No. 5,556,752, Lockhart et al., entitled“Surface-bound, unimolecular, double-stranded DNA,” issued Sep. 17,1996; and U.S. Pat. No. 5,510,270, Fodor et al., entitled “Synthesis andscreening of immobilized oligonucleotide arrays,” issued Apr. 23, 1996;each of which is incorporated by reference in its entirety for allpurposes) or other methods for rapid synthesis and deposition of definedoligonucleotides (Lipshutz et al., 1999, Nat. Genet. 21(1 Suppl):20-4.)

When these methods are used, oligonucleotides (e.g., 20-mers) of knownsequence are synthesized directly on a surface such as a derivatizedglass slide. Usually, the array produced contains multiple probesagainst each target transcript. Oligonucleotide probes can be chosen todetect alternatively spliced mRNAs or to serve as various type ofcontrol.

In a particularly preferred embodiment, microarrays of the invention aremanufactured by means of an ink jet printing device for oligonucleotidesynthesis, e.g., using the methods and systems described by Blanchard inInternational Patent Publication No. WO 98/41531, published Sep. 24,1998; Blanchard et al., 1996, Biosensors and Bioele[c?]tronics11:687-690; Blanchard, 1998, in Synthetic DNA Arrays in GeneticEngineering, Vol. 20, J. K. Setlow, Ed., Plenum Press, New York at pages111-123; U.S. Pat. No. 6,028,189 to Blanchard.

Specifically, the oligonucleotide probes in such microarrays arepreferably synthesized in arrays, e.g., on a glass slide, by seriallydepositing individual nucleotide bases in “microdroplets” of a highsurface tension solvent such as propylene carbonate. The microdropletshave small volumes (e.g., 100 pL or less, more preferably 50 pL or less)and are separated from each other on the microarray (e.g., byhydrophobic domains) to form circular surface tension wells which definethe locations of the array elements (i.e., the different probes).

Other methods for making microarrays, e.g., by masking (Maskos andSouthern, 1992, Nuc. Acids Res. 20:1679-1684), may also be used. Inprincipal, any type of array, for example, dot blots on a nylonhybridization membrane (see Sambrook et al., 1989, Molecular Cloning—ALaboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.), could be used, although, as will berecognized by those of skill in the art, very small arrays will bepreferred because hybridization volumes will be smaller.

5.6.5. Hybridization to Microarrays

Nucleic acid hybridization and wash conditions are optimally chosen sothat the probe “specifically binds” or “specifically hybridizes” to aspecific array site, i.e., the probe hybridizes, duplexes or binds to asequence array site with a complementary nucleic acid sequence but doesnot hybridize to a site with a non-complementary nucleic acid sequence.As used herein, one polynucleotide sequence is considered complementaryto another when, if the shorter of the polynucleotides is less than orequal to 25 bases, there are no mismatches using standard base-pairingrules or, if the shorter of the polynucleotides is longer than 25 bases,there is no more than a 5% mismatch. Preferably, the polynucleotides areperfectly complementary (no mismatches). It can easily be demonstratedthat specific hybridization conditions result in specific hybridizationby carrying out a hybridization assay including negative controls (see,e.g., Shalon et al., 1996, Genome Research 6:639-645, and Chee et al.,1996, Science 274:610-614).

Optimal hybridization conditions will depend on the length (e.g.,oligomer versus polynucleotide greater than 200 bases) and type (e.g.,RNA, DNA, PNA) of labeled probe and immobilized polynucleotide oroligonucleotide. General parameters for specific (i.e., stringent)hybridization conditions for nucleic acids are described in Sambrook etal. (1989, Molecular Cloning—A Laboratory Manual (2nd Ed.), Vols. 1-3,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.) and in Ausubelet al. (1987, Current Protocols in Molecular Biology, Greene Publishing,Media, Pa., and Wiley-Interscience, New York). When the cDNA microarraysof Schena et al. (1996, Proc. Natl. Acad. Sci. USA, 93:10614-19) areused, typical hybridization conditions are hybridization in 5×SSC plus0.2% SDS at 65° C. for 4 hours followed by washes at 25° C. in lowstringency wash buffer (1×SSC plus 0.2% SDS) followed by 10 minutes at25° C. in high stringency wash buffer (0.1×SSC plus 0.2% SDS) (Schena etal., 1996, Proc. Natl. Acad. Sci. USA, 93:10614-19). Usefulhybridization conditions are also provided in, e.g., Tijssen, 1993,Hybridization With Nucleic Acid Probes, Elsevier Science PublishersB.V., Amsterdam and New York, and Kricka, 1992, Nonisotopic DNA ProbeTechniques, Academic Press, San Diego, Calif.

Although simultaneous hybridization of differentially labeled mRNAsamples is preferred, it is also possible to use a single label and toperform hybridizations sequentially rather than simultaneously.

5.6.6. Signal Detection and Data Analysis

When fluorescently labeled probes are used, the fluorescence emissionsat each site of a transcript array can be, preferably, detected byscanning confocal laser microscopy. In one embodiment, a separate scan,using the appropriate excitation line, is carried out for each of thetwo fluorophores used. Alternatively, a laser can be used that allowssimultaneous specimen illumination at wavelengths specific to the twofluorophores and emissions from the two fluorophores can be analyzedsimultaneously (see Shalon et al., 1996, Genome Research 6:639-645,which is incorporated by reference in its entirety for all purposes). Ina preferred embodiment, the arrays are scanned with a laser fluorescentscanner with a computer controlled X-Y stage and a microscope objective.Sequential excitation of the two fluorophores is achieved with amulti-line, mixed gas laser and the emitted light is split by wavelengthand detected with two photomultiplier tubes. Fluorescence laser scanningdevices are described in Shalon et al., 1996, Genome Res. 6:639-645 andin other references cited herein. Alternatively, the fiber-optic bundledescribed by Ferguson et al., 1996, Nature Biotechnol. 14:1681-1684, maybe used to monitor mRNA abundance levels at a large number of sitessimultaneously.

Signals are recorded and, in a preferred embodiment, analyzed bycomputer, e.g., using a 12 bit analog to digital board. In oneembodiment the scanned image is bespeckled using a graphics program(e.g., Hijaak Graphics Suite) and then analyzed using an image griddingprogram that creates a spreadsheet of the average hybridization at eachwavelength at each site. If necessary, an experimentally determinedcorrection for “cross talk” (or overlap) between the channels for thetwo fluors may be made. For any particular hybridization site on thetranscript array, a ratio of the emission of the two fluorophores can becalculated. The ratio is independent of the absolute expression level ofthe cognate gene, but is useful for genes whose expression issignificantly modulated by drug administration, gene deletion, or anyother tested event.

According to the method of the invention, the relative abundance of anmRNA in two biological samples is scored as a perturbation and itsmagnitude determined (i.e., the abundance is different in the twosources of mRNA tested), or as not perturbed (i.e., the relativeabundance is the same). In various embodiments, a difference between thetwo sources of RNA of at least a factor of about 25% (RNA from onesource is 25% more abundant in one source than the other source), moreusually about 50%, even more often by a factor of about 2 (twice asabundant), 3 (three times as abundant) or 5 (five times as abundant) isscored as a perturbation.

Preferably, in addition to identifying a perturbation as positive ornegative, it is advantageous to determine the magnitude of theperturbation. This can be carried out, as noted above, by calculatingthe ratio of the emission of the two fluorophores used for differentiallabeling, or by analogous methods that will be readily apparent to thoseof skill in the art.

In one embodiment, two samples, each labeled with a different fluor, arehybridized simultaneously to permit differential expressionmeasurements. If neither sample hybridizes to a given spot in the array,no fluorescence will be seen. If only one hybridizes to a given spot,the color of the resulting fluorescence will correspond to that of thefluor used to label the hybridizing sample (for example, green if thesample was labeled with Cy3, or red, if the sample was labeled withCy5). If both samples hybridize to the same spot, an intermediate coloris produced (for example, yellow if the samples were labeled withfluorescein and rhodamine). Then, applying methods of patternrecognition and data analysis known in the art, it is possible toquantify differences in gene expression between the samples. Methods ofpattern recognition and data analysis are described in e.g., co-pendingU.S. patent application Ser. No. 09/179,569 filed on Oct. 27, 1998, byFriend et al.; Ser. No. 09/220,142 filed on Dec. 23, 1998, by Stoughtonet al.; Ser. No. 09/220,275 filed on Dec. 23, 1998, by Friend et al.;International Publication WO 00/24936, dated May 4, 2000, which areincorporated by reference herein in their entireties.

5.7. Diagnostic Methods

The random-primed RT-IVT methods of the invention have use in nucleicacid amplification reactions to generate sufficient quantities ofnucleic acid for detection of a specific nucleic acid of interest.Accordingly, the methods of the invention can be used in methods ofdiagnosis, for example, in amplifying a sequence (e.g., genomic) of aninfectious disease agent, e.g., of human disease including but notlimited to viruses, bacteria, parasites, and fungi, thereby diagnosingthe presence of the infectious agent in a sample of nucleic acid from apatient. The nucleic acid of interest can be genomic or cDNA or mRNA, orcan be synthetic, human or animal, or of a microorganism, etc. Inanother embodiment that can be used in the diagnosis or prognosis of adisease or disorder, the nucleic acid of interest is a wild type humangenomic or RNA or cDNA sequence, mutation of which is implicated in thepresence of a human disease or disorder, or alternatively, can be themutated sequence. By way of example, the mutation can be an insertion,substitution, and/or deletion of one or more nucleotides, or atranslocation.

5.8. Kits for the Amplification and Detection of Selected TargetNucleotide Sequences

The present invention also provides kits for the linear amplification ofRNA, and, for example, detection or measurement of nucleic acidamplification products and for determining the responses or state of abiological sample. Such a kit may comprise containers, each with one ormore of the various reagents (typically in concentrated form) utilizedin the methods of the invention, including, for example, buffers, theappropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP,CTP, GTP and UTP), reverse transcriptase, RNA polymerase specific to theRNA polymerase promoter, and the random promoter-primers and primers ofthe present invention. Optionally also present in the kit is a reversetranscriptase inhibitor, where, in many embodiments, the inhibitor is atleast ddNTP or a combination of ddNTPs, e.g., ddATP and/or ddGTP. A setof instructions for use of kit components in an mRNA amplificationmethod of the present invention, will also be typically included.

In a specific embodiment, the kit comprises one or more primeroligonucleotides of the invention, such as a RNA polymerasepromoter-containing primer, including but not limited to a set of randomRNA polymerase promoter-containing primers and/or a set of randomprimers, in one or more containers. The kit can comprise for example, arandom T7-poly dN primer set, a T7-poly dT primer, and/or a random polydN primer set. The kit can further comprise additional components forcarrying out the amplification reactions of the invention, such asreverse transcriptase and RNA polymerase. Where the target nucleic acidsequence being amplified is one implicated in disease or disorder, thekit can he used for diagnosis or prognosis.

Oligonucleotides in containers can be in any form, e.g., lyophilized, orin solution (e.g., a distilled water or buffered solution), etc.Oligonucleotides ready for use in the same amplification reaction can becombined in a single container or can be in separate containers.

The kit optionally further comprises a control nucleic acid, and/or amicroarray, and/or means for stimulating and detecting fluorescent lightemissions from fluorescently labeled RNA, and/or expression profileprojection and analysis software capable of being loaded into the memoryof a computer system. The kit optionally further provides means forstimulating and detecting fluorescent light emissions, e.g., afluorescence plate reader or a combination thermocycler-plate-reader toperform the analysis.

5.8.1. Analytic Kit Implementation

In a preferred embodiment, the methods of this invention can beimplemented by use of kits containing oligonucleotide primers of theinvention and microarrays. The microarrays contained in such kitscomprise a solid phase, e.g., a surface, to which probes are hybridizedor bound at a known location of the solid phase. Preferably, theseprobes consist of nucleic acids of known, different sequence, with eachnucleic acid being capable of hybridizing to a RNA species or to a cDNAspecies derived therefrom. In particular, the probes contained in thekits of this invention are nucleic acids capable of hybridizingspecifically to nucleic acid sequences derived from RNA species that areknown to increase or decrease in response to perturbations to theparticular protein whose activity is determined by the kit. The probescontained in the kits of this invention preferably substantially excludenucleic acids that hybridize to RNA species that are not increased inresponse to perturbations to the particular protein whose activity isdetermined by the kit.

In another preferred embodiment, a kit of the invention further containsexpression profile projection and analysis software capable of beingloaded into the memory of a computer system. An example of such a systemis described in co-pending U.S. patent application Ser. No. 09/220,276,by Bassett, Jr. et al., filed Dec. 23, 1998, which is incorporatedherein by reference in its entirety. Preferably, the expression profileanalysis software contained in a kit of this invention, is essentiallyidentical to the expression profile analysis software 512 described inU.S. patent application Ser. No. 09/220,276.

Alternative kits for implementing the analytic methods of this inventionwill be apparent to one of skill in the art and are intended to becomprehended within the accompanying claims. In particular, theaccompanying claims are intended to include the alternative programstructures for implementing the methods of this invention that will bereadily apparent to one of skill in the art.

The following experimental examples are offered by way of illustrationand not by way of limitation.

6. EXAMPLE 1 Cdna Synthesis and RNA Amplification for the Preparation ofCy3- and Cy5-Labeled RNA Targets for Gene Expression Monitoring

This example demonstrates that using the random-primed RT-IVT method ofthe invention, linear amplification of mRNA to[<CUT?] can be used toproduce unbiased antisense RNA profiles. The results of an mRNAamplification produced using the random-primed RT-IVT method of theinvention were compared with results obtained using the mRNAamplification method disclosed in Shannon (U.S. Pat. No. 6,132,997,entitled “Method for linear mRNA amplification,” issued Oct. 17, 2000).Using the random primed RT-IVT method, poly-A+RNA was converted todouble-stranded cDNA using degenerate random primers comprising a T7 RNApolymerase promoter sequence (T7-dN₉) to prime first strand cDNAsynthesis and degenerate random primers (dN₆) to prime second strandcDNA synthesis to yield a double-stranded cDNA that is recognized by T7RNA polymerase. The double-stranded cDNA was then transcribed intoantisense RNA by T7 RNA polymerase in the presence of a reversetranscriptase that was rendered incapable of RNA-dependent DNApolymerase activity during this transcription step by heat inactivation.5-(3-Aminoallyl)uridine 5′-triphosphate was incorporated into theantisense RNA during transcription and post-synthetically labeled withCy3-NHS or Cy5-NHS. Linear amplification extents of at least 100-foldand labeling efficiencies of approximately 3% were achieved using thismethod.

6.1. Materials and Methods

Total RNA was isolated from Jurkat and K562 cell lines. Poly-A⁺ RNA wasisolated from the total RNA to provide the initial source mRNA used inthe experiment.

cDNA Synthesis Reagents.

-   -   1. mRNA, 0.2 mg.    -   2.

(SEQ ID NO.: 1) DNA T7-dN₉ (20 μM): (5′) AAT TAA TAC GAC TCA CTATAG GGA GAT NNN NNN NNN (3′) (N = A, T, C or G)

-   -   3. MMLV Reverse Transcriptase (50 U/μl), Epicentre P/N M4425H    -   4. RNAGuard™, Pharmacia P/N 27-0815-01    -   5. 5× First Strand Buffer: 250 mM Tris-HCl, pH 8.3, 15 MM MgCl₂,        375 mM KCl, Life Technologies P/N 18057-018    -   6. 100 mM DTT* (*supplied with MMLV Reverse Transcriptase,        Epicentre)    -   7. dNTPs (10 mM each), diluted from Pharmacia P/N 2702035-01    -   8. ultraPURE distilled water, DNAse, RNAse Free, Life        Technologies, Cat #10977-015    -   9. pdN₆ (200 ng/μl), diluted from Amersham Pharmacia Biotech P/N        27-2166-01 Transcription Reagents:    -   1. T7 RNA Polymerase (2500units/A, Epicentre P/N TU950K    -   2. RNAGuard™, Pharmacia P/N 27-0815-01    -   3. Inorganic Pyrophosphatase (200 U/ml), New England Biolabs,        #M0296S    -   4. 5× Transcription Buffer: 0.2 M Tris-HCl, pH 7.5, 50 mM NaCl,        30 MM MgCl₂, 10 mM spermidine, Epicentre P/N BP1001    -   5. 100 mM DTT, Epicentre P/N BP1001    -   6. MgCl₂ (200 mM), diluted from Sigma P/N M-1028    -   7. NTPs (25 mM ATP, GTP, CTP, 6 mM UTP), diluted from Pharmacia        P/N 27-2025-01    -   8. 5-(3-Aminoallyl)uridine 5′-triphosphate (75 mM), Sigma P/N        A-5660    -   9. ultraPURE distilled water, DNAse, RNAse Free, Life        Technologies, P/N 10977-015

Purification and Labeling Reagents:

-   -   1. RNeasy® Mini Kit (250), QIAGEN Inc., P/N 74106    -   2. Carbonate-Bicarbonate Buffer capsules, Sigma, P/N C-3041    -   3. Hydrochloric acid, Fisher, P/N A508-500    -   4. Anhydrous MSO (methyl sulfoxide, also known as DMSO, dimethyl        sulfoxide), Aldrich, P/N 27,685-5    -   5. Cy3-NHS dye pack, Amersham, P/N PA23001    -   6. Cy5-NHS dye pack, Amersham, P/N PA25001    -   7. Hydroxylamine (“HA”), Sigma, P/N H-2391        Other materials:    -   1. Pipetman micropipettors, (P-10, P-20, P-200, P-1000), or        equivalent    -   2. Sterile, nuclease-free 1.5 ml microcentrifuge tubes    -   3. Sterile, nuclease-free aerosol-barrier pipet tips    -   4. Thermal Cycler

Reagent Preparation:

-   -   1. dNTPs (10 mM each)    -   Thaw dNTP stocks (100 mM) and place on ice. Add 10 μl each dNTP        to 60 μl nuclease-free water. Store frozen.    -   2. pdN₆ (200 ng/μl)    -   Add 663 μl nuclease-free water to lyophilized sample (50 A260        units or approximately 1325 μg) for 2.0 μg/μl. Add 10 μl pdN₆        (2.0 μg/μl) to 90 μl nuclease-free water for 200 ng/μl. Store        frozen.    -   3. 200 mM MgCl₂    -   Add 100 μl of 1 M MgCl₂ to 400 μl nuclease-free water. Store        frozen.    -   4. NTPs (25 mM ATP, GTP, CTP, 6.0 mM UTP)    -   Thaw NTP stocks (100 mM) and place on ice. Combine 125 μl ATP,        125 μl GTP, 125 μl CTP, 30 μl UTP and 95 μl nuclease-free water.        Store frozen.    -   5. aa UTP (75 mM) Dissolve 5 mg in 125 μl water.    -   6. Anhydrous MSO should be stored with a molecular sieve to        absorb water.

Procedure:

To prevent contamination of reactions by ribonucleases, laboratorygloves were worn and dedicated solutions and pipettors withnuclease-free, aerosol-resistant tips were used.

Amplified RNA preparations were prepared in batches of no less than 6 tominimize errors associated with pipetting small volumes of enzymesolutions. The procedure below specifies reagent volumes for 1 reaction;for 6 reactions, the specified volumes were multiplied by 6.5.

1. Add 0.2 μg of source mRNA to reaction tube. Add 1.0 μl DNA T7-dN₉ (20μM) and bring total sample volume to 10.5 μl in nuclease-free water.

2. Incubate at 65° C. for 10 min to denature primer and template. Movereaction tubes to ice. Store reactions tubes on ice for 5 rain.

3. Mix the following components and maintain on ice.

cDNA Mix Component Volume (μl) 5x First Strand Buffer 4.0 100 mM DTT 2.0dNTPs (10 mM each) 1.0 pdN₆ (200 ng/μl) 1.0 MMLV-RT (50 U/μl) 1.0RNAGuard ™ (36 U/μl) 0.5 Volume of cDNA Mix 9.5

4. Aliquot 9.5 μl of cDNA Mix into each sample tube. Incubate cDNAsynthesis reaction at 40° C. for 120 min.

Composition of cDNA Synthesis Reaction Final concentration Component oramount poly-A⁺ RNA 200 ng DNA T7T18VN 1 μM Tris-HCl, pH 8.3 50 mM MgCl₂3.0 mM KC1 75 mM DTT 10 mM dNTPs mM each MMLV-RT 50 U RNAGuard ™ 18 UTotal reaction volume 20 μl

Incubate reaction tubes at 65° C. for 15 min. This inactivates thereverse transcriptase activity of MMLV prior to the IVT step. Movereaction tubes to ice. Store reaction tubes on ice for 5 min.

5. Immediately before use, mix the following components in the orderindicated at room temperature:

Transcription Mix Component Volume (μl) Nuclease-free water 22.8 5xTranscription Buffer 16 100 mM DTT 6.0 NTPs (25 mM A, G, C, 6.0 mM UTP)8.0 aa UTP (75 mM) 2.0 200 mM MgCl₂ 3.3 RNAGuard ™ (36 U/μl) 0.5Inorganic Pyrophosphatase (200 U/ml) 0.6 T7 RNA polymerase (2500 U/μl)0.8 Volume of Transcription Mix 60

6. Aliquot 60 μl of Transcription Mix into each sample tube. Incubatetranscription reactions at 40° C. for 16 hrs.

Composition of Transcription Reaction Final concentration Component oramount Double-strand cDNA Approximately 400 ng Tris-HCl, pH 7.5 52 mMMgCl₂ 15 mM KCl 19 mM NaCl 10 mM Spermidine 2 mM DTT 10 mM ATP, GTP, CTP2.5 mM each UTP 0.6 mM aa UTP 1.9 mM T7 RNA polymerase 2000 U RNAGuard ™18 U Inorganic pyrophosphatase 0.12 U Total reaction volume 80 μl

7. RNeasy® (QIAGEN Inc.) Purification of reactions:

-   -   Add 20 μl water to 80 μl reaction tube.    -   Transfer to mixing tube.    -   Add 350 μl RLT buffer (QIAGEN Inc.) (plus 2-β-mercaptoethanol),        mix well.    -   Add 250 μl 100% EtOH, mix well.    -   Transfer to RNeasy® column.    -   Spin 30 seconds in microfuge, 10 K.    -   Transfer column to new collection tube.    -   Add 700 W 80% EtOH.    -   Spin 30 seconds in microfuge, 10 K.    -   Discard flow through.    -   Add 700 μl 80% EtOH.    -   Spin 30 seconds in microfuge, 10 K.    -   Transfer column to new collection tube.    -   Spin 2 minutes, 14 K to dry filter.    -   Place column in microfuge tube.    -   Add 55 μl of nuclease-free water to filter. Let sit 1 minute.    -   Spin 14 K, 2 minutes.    -   Add 55 μl of nuclease-free water to filter. Let sit 1 minute.    -   Spin 14 K, 2 minutes.

To quantitate the yield of amplified RNA product, remove a 10.0 μlaliquot of the product and dilute into 90 μl dH₂O. Add samples to aCostar UV-transparent plate and measure A260, A280 using a Spectramax(GRM Reader) and template for whichever lot of plates you are using.Calculate yield using the relationship A260=1 corresponds to 40 μg/ml.Conversion factor for Spectramax=3.59 (i.e. multiply A260 by 3.59 whencalculating yield).

8. In speed vac, dry down 10 μg per fluor-reversed pair.

9. Coupling Reactions:

-   -   Resuspend 10 μg IVT product in 7 μl water (or water plus El a)        and divide into two tubes. One tube will be coupled with Cy3 and        one with Cy5.

Preparation of 3× Sodium Bicarbonate Buffer:

Place the contents of one Carbonate-Bicarbonate Buffer capsule (Sigma,P/N C-3041) into a 50 ml Falcon tube.

Add 16.7 ml RNase free water and mix well.

Add 125 μl 37% HCl and mix.

pH should be 9- 9.5.

Preparation of Cy-NHS dyes:

Spin dye briefly before opening tube.

Add 10 μl anhydrous MSO to dye.

Mix by pipetting 20 times.

Set a pipettman at 3.5 μl.

Work quickly since the amino esters are unstable in aqueous environment.

Add 20 μl 3× sodium bicarbonate buffer to dye and mix well.

Add 3.5 μl dye to each tube of cRNA. Mix well.

Incubate in the dark for 1 hour.

Stop the reaction by adding 3.5 μl 4M HA (hydroxylamine)

Incubate 10 minutes.

10. Repeat RNeasy® clean-up as in Step 7, above, except elute in 70° C.nuclease-free water.

11. Measure yield and percent incorporation in a Costar UV plate.Calculate concentration of RNA using 1 OD₂₆₀=40 μg/ml RNA. Overallamplification yield is calculated by multiplying RNA concentration(μg/ml) by the sample volume (0.1 ml) and dividing by the amount ofpoly-A⁺ RNA initially added to the reaction. Calculate concentration ofCy3-CTP using ε(552 nm)=150 (1/mMcm). Calculate concentration of Cy5-CTPusing ε(650 nm)=250 (1/mMcm).

Generation of Gene Expression Profile Signatures:

Source mRNA from Jurkat and K562 cell lines was used to generate geneexpression profile signatures by amplification and labeling using theShannon method and using the random-primed RT-IVT method, followed byhybridization to DNA microarrays. Approximately 5 μg of Cy-labeled cRNAfrom each cell line was hybridized as fluor-reversed pairs to a DNAmicroarray pattern with probes tiled (overlapped) across all mRNAsequence for approximately 33 RefSeq test genes (LocusLink database,www.ncbi.nlm.nih gov/locuslink/build.html) known to exhibit 3′amplification bias when amplified by the Shannon method. Analysis wasperformed either on a gene-by-gene basis or with all oligonucleotides atonce. The first goal of the study was to determine whether therandom-primed RT-IVT method produced a full-length cRNA. The second goalof the study was to determine whether the random-primed RT-IVT methodhas less of a 3′ bias when compared with the Shannon method.

6.2. Results and Discussion

FIG. 1 compares the profiles obtained from single-gene analysis usingthe mRNA amplification method described in U.S. Pat. No. 6,132,997(Shannon, issued Oct. 17, 2000) (“Shannon”) and the random-primed RT-IVTmethod of the invention. The graphs plot signal intensity (mlavg) ofoligonucleotides in a single gene (X-axis) as a function of the numberof by from the 5′ end (Y-axis). The 3′ bias of signal intensity seenwhen the Shannon method is used cannot be seen when the random-primedRT-IVT method is used, indicating that the random-primed RT-IVT methodovercomes the 3′ bias of the Shannon method.

FIG. 2 shows the intensity difference as a function of distance from the3′ end. The graph shows the intensity of all oligonucleotides as afunction of distance from the 3′ end.

The graph plots mlavg (Shannon method) - mlavg (random-primed RT-IVTmethod) (X-axis) versus log₁₀ of the number of by from the 3′ end(Y-axis). The intensity obtained with the Shannon method is greater thanthe intensity obtained with the random-primed RT-IVT method for probesless than 1000 by from the 3′ end of the message. The intensity obtainedwith the Shannon method is less than the intensity obtained with therandom-primed RT-IVT method for probes greater than 1000 by from the 3′end of the message.

At xdev threshold 2.5 (˜Pvalue 1%), the following number of signatureswere obtained (Table 2):

TABLE 2 Forward Reverse by < bp > by < bp > Total 1000 1000 total 10001000 random-primed 2486 1488 998 2237 1367 870 RT-IVT Shannon method2587 1927 660 1218 892 326 # of probes 7416 2965 4451 7413 2962 4451

FIGS. 3(A-C) shows[show?] the signature differences in the numbers andpercentages of significant data points. The top graph (A) plots thenumber of probes (X-axis) versus the [log₁₀] (bp) (Y-axis). The middlegraph (B) plots the number of signatures (X-axis) versus the [log₁₀](bp) (Y-axis). The bottom graph (C) plots the fraction of signaturesversus the [log₁₀] (bp) (Y-axis). As can be seen in the bottom graph,the random-primed RT-IVT method outcompetes the Shannon method forprobes greater than 1000 by from the 3′ end. Note the black arrow atapproximately 700 by where random-primed RT-IVT method becomes betterthan the Shannon method. Stars: Shannon method. Circles: random-primedRT-IVT method.

FIGS. 4(A-C) shows the results obtained when the amplification methodsof the invention were run using a primer comprising a T7 RNA polymerasepromoter site and a poly-dT₁₈ sequence (“T7-dT₁₈”), in addition to usingrandom T7-d N₉ and dN₆ primers. The top graph (A) plots the number ofprobes (X-axis) versus the log₁₀ (bp) (Y-axis). The middle graph (B)plots the number of signatures (X-axis) versus the log₁₀ (bp) (Y-axis).The bottom graph (C) plots the fraction of signatures versus the log₁₀(bp) (Y-axis). As can be seen in the bottom graph, the random-primedRT-IVT method helps improve the fraction of significant probes at by<1000. Using both the T7-dT₁₈ and random T7-dN₉ primers for first strandcDNA synthesis improves the fraction of significant probes moreefficiently than either the Shannon method or the method of theinvention in which just the random T7-d N₉ primer is used. Stars:Shannon method. Circles: random-primed RT-IVT method.

These results indicate that the performance of random-primed RT-IVT isstable. The average yield obtained was 20 μg. The protocol producedlittle or no 3′ bias and improved the ability to detect the 5′ ends ofmRNA. Linear amplification extents of 100-fold and labeling efficienciesof approximately 3% can be achieved using this method. When poly-dT andrandom dN primers, both of which comprise a T7 RNA polymerase promotersequence, are used together to prime first strand cDNA synthesis, thefraction of significant probes is greater than that obtained with eitherthe Shannon method or the method of the invention in which just a randomT7-dN₉ primer is used.

The above results and discussion demonstrate that novel and improvedmethods of producing linearly amplified amounts of RNA from an initialRNA source are provided. The methods of the invention provide animprovement over prior methods of producing linearly amplified RNA inthat the protocol produces little or no 3′ bias and improves the abilityto detect the 5′ ends of mRNA. Furthermore, linear amplification extentsof at least 100-fold can be achieved using the subject methods. Finally,all of the benefits of linear amplification are achieved with thesubject methods, such as the production of unbiased antisense RNAlibraries from heterogeneous mRNA mixtures. As such, the subject methodsrepresent a significant contribution to the art.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The citation of any publication is for its disclosure prior to thefiling date and should not be construed as an admission that the presentinvention is not entitled to antedate such publication by virtue ofprior invention.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims along with the full scope ofequivalents to which such claims are entitled.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

1. A method for amplifying one or more single stranded nucleic acids,said method comprising: (a) contacting said one or more single strandednucleic acids with a first set of oligonucleotides, each of saidoligonucleotides in said first set comprising a promoter sequence and asequence from a set of random sequences of at least 4 nucleotides, asecond set of oligonucleotides, each of said oligonucleotides in saidsecond set comprising one of a set of random sequences of at least fournucleotides and one or more enzymes that alone or in combinationcatalyze the synthesis of double-stranded cDNA, under-conditionssuitable for the production of double-stranded cDNA; and (b) contactingthe double-stranded cDNA produced in, step (a) with a RNA polymerasethat recognizes said promoter sequence and ribonucleotides underconditions suitable to effect transcription, thereby producing sense orantisense RNA copies corresponding to said one or more single strandednucleic acids.
 2. The method of claim 1, wherein the one or more singlestranded nucleic acids are poly-A+ RNA.
 3. The method of claim 1,wherein the one or more enzymes is a reverse transcriptase.
 4. Themethod of claim 3, wherein the reverse transcriptase is renderedincapable of RNA-dependent DNA polymerase activity during thetranscription step.
 5. The method of claim 4, wherein prior to step (b)said reverse transcriptase is inactivated. 6-7. (canceled)
 8. The methodof claim 1, wherein the random sequences of the oligonucleotides in saidfirst set are 6 to 9 nucleotides.
 9. The method of claim 1, wherein therandom sequences of the oligonucleotides in said second set are 6 to 9nucleotides.
 10. The method of claim 1, wherein the random sequences ofthe oligonucleotides in said first set are 9 nucleotides.
 11. The methodof claim 1, wherein the random sequences of the oligonucleotides in saidsecond set are 6 nucleotides.
 12. The method of claim 1, wherein theoligonucleotides in said second set do not comprise a promoter sequence.13. (canceled)
 14. The method of claim 1, wherein step (a) furthercomprises contacting said one or more single-stranded nucleic acids witha third set of oligonucleotides each of said oligonucleotides of saidthird set comprising the promoter sequence and a polydT sequence of atleast 5 nucleotides.
 15. The method of claim 14, wherein said polydTsequence is 5 to 25 nucleotides.
 16. The method of claim 15, whereinsaid polydT sequence is 18 nucleotides.
 17. The method of claim 1,wherein the promoter sequence is a T7 RNA polymerase promoter sequenceand the RNA polymerase is T7 RNA polymerase.
 18. The method of claim 1,wherein the ribonucleotides comprise 5-(3-Aminoallyl)uridine5′-triphosphate.
 19. The method of claim 1, wherein the sense orantisense RNA copies are labeled with Cy-NHS.
 20. The method of claim19, wherein the Cy-NHS is Cy3-NHS or Cy5-NHS.
 21. A kit for use inamplifying single stranded nucleic acids into sense or antisense RNA,said kit comprising in one or more containers a first set ofoligonucleotides, each of said oligonucleotides in said first setcomprising a promoter sequence and one of a set of random sequences ofat least 4 nucleotides; and a -second set of oligonucleotides, each ofsaid oligonucleotides in said second set comprising one of a set ofrandom sequences of at least four nucleotides.
 22. The kit of claim 21,which further comprises a reverse transcriptase and a RNA polymerasethat recognizes said promoter sequence. 23-31. (canceled)
 32. A methodfor amplifying one or more single stranded nucleic acids, said methodcomprising: (a) contacting said one or more single stranded nucleicacids with a first set of oligonucleotides, each of saidoligonucleotides in said first set comprising a promoter sequence and asequence from a set of random sequences of at least 4 nucleotides, andone or more enzymes that catalyze the synthesis of first strand cDNA,under conditions suitable for the production of first strand cDNA; (b)contacting the first strand cDNA produced In step (a) with a second setof oligonucleotides, each of said oligonucleotides in said second setcomprising one of a set of random sequences of at least four nucleotidesand one or more enzymes that catalyze the synthesis of double-strandedcDNA, under conditions suitable for the production of double-strandedcDNA; and (c) contacting the double-stranded cDNA produced in step (b)with a RNA polymerase that recognizes said promoter sequence-andribonucleotides under conditions suitable to effect transcription,thereby producing sense or antisense RNA copies corresponding to saidone or more single stranded nucleic acids.